Compositions and methods for the treatment of progressive multifocal leukoencephalopathy (pml)

ABSTRACT

The invention relates to compositions, methods, and kits for treating subjects infected by or at risk of infection with a DNA virus (e.g., a JC Virus or a BK virus). Aspects of the invention are useful to prevent or treat DNA virus associated conditions (e.g., PML) in subjects that are immuno-compromised. Compositions are provided that inhibit intracellular replication of DNA viruses.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/678,011, filed Oct. 7, 2010, which is a national stage filing under35 U.S.C. §371 of PCT International application PCT/US2008/010734designating the United States of America, and filed Sep. 13, 2008, whichclaims the benefit under 35 U.S.C. §119(e) of U.S. provisionalapplication Ser. No. 60/993,769, filed Sep. 14, 2007, the entirecontents of each of which are herein incorporated by reference.

FIELD OF THE INVENTION

The invention relates to compositions and methods for treating subjectsinfected with a DNA virus. In particular, aspects of the inventionrelate to subjects infected with JCV virus and subjects having, or atrisk for developing, progressive multifocal leukoencephalopathy (PML).

BACKGROUND OF THE INVENTION

JC polyomavirus (JCV) is the causative agent of a demyelinating diseaseof the central nervous system, progressive multifocalleukoencephalopathy (PML). The incidence of PML can be related to aweakened immune system or treatment with immunosuppressants. Currently,there is no specific antiviral therapy that has been proven effectivefor treatment of PML.

SUMMARY OF THE INVENTION

Aspects of the invention relate to compositions that inhibit DNA virusactivity, including viral proliferation (e.g., viral replication),mutation rate and infectivity, and the use of such compositions to treator suppress conditions associated with DNA virus activity in subjectsthat are infected with a DNA virus, or the use of such compositions tolower the risk of infection with the DNA virus. In some embodiments, theinvention provides one or more compositions that inhibit JCV activity.In some embodiments, the invention provides one or more compositionsthat inhibit BK virus (BKV) activity. Such compositions may be used toprevent DNA viral infection (e.g., JCV infection or BKV infection), toprevent an increase in DNA viral activity (e.g., active JCV infection ofthe brain), to prevent DNA virus proliferation (e.g., JCV proliferationor BKV proliferation) to prevent symptoms associated with viralinfection (e.g., JCV or BKV infection), to treat a subject infected witha DNA virus (e.g., JCV or BKV), or treat a subject at risk of infectionwith a DNA virus (e.g., JCV or BKV), or to treat a subject that hasdeveloped a disease or condition associated with infection by a DNAvirus (e.g., PML). Compositions of the invention also may beadministered to a subject at risk of a viral infection or at risk of anincrease in viral activity (e.g., viral proliferation, for example inthe brain or CNS), regardless of whether the subject is actually knownto have been exposed to, or infected by, the virus.

In some embodiments, one or more compositions of the invention may beadministered to subjects that have a compromised immune system. Itshould be appreciated that a subject's immune system may be compromiseddue to treatment with an immunosuppressive therapeutic agent and/or dueto a disease or condition that impacts the immune system. In someembodiments, one or more compositions of the invention may beadministered to a subject that is at risk of PML due to a compromisedimmune system, regardless of whether the subject is known to be infectedwith JCV or known to have been exposed to JCV. Accordingly, compositionsof the invention may be administered to subjects that are receiving animmunosuppressive treatment for a disease or condition. In someembodiments, compositions of the invention may be administered tomultiple sclerosis (MS) patients that are being treated with one or moreimmunosuppressive agents (e.g., natalizumab). However, in someembodiments, compositions of the invention may be administered tosubjects that have a weakened immune system caused by a disease orcondition itself, rather than by an immunosuppressive treatment. Forexample, subjects infected with an immuno-compromising pathogen (e.g., avirus such as HIV) may be treated with one or more compositions of theinvention.

It should be appreciated that while the JCV status of a subject need notbe known, it may be useful to know the status in some embodiments. Insome embodiments, the efficacy of such treatment or therapy may bemonitored by detecting and/or monitoring the presence of JCV in asubject.

In some embodiments, one or more compositions of the invention may beadministered to a subject before, during, and/or after the subjectreceives and immunomodulatory therapy (e.g., a treatment that inhibitsthe immune system of the subject). Accordingly, in some embodiments oneor more compounds described herein as being effective to inhibit DNAvirus replication may be administered to a subject prior to initiationof an immunomodulatory therapy. For example, a therapeutic regimen ofone or more compositions of the invention may be initiated prior to animmunomodulatory treatment against a disease or in preparation for atransplant in to prevent or reduce any risk of DNA virus replication orproliferation associated with the immunomodulatory treatment.

In some embodiments, one or more compositions of the invention may beadministered alone or in combination with other compositions describedherein or along with other therapeutic agents (e.g., one or moreimmunosuppressive therapeutic agents). Compositions of the invention maybe provided (e.g., administered) in pharmaceutical preparations.Compositions of the invention may be provided in kits.

In some aspects, compositions of the invention may be used to developfurther anti-viral treatments (e.g., as starting material for amedicinal chemistry study or as references in an in vitro assay).

In some aspects, the invention provides methods of inhibiting viralreplication, the methods comprising contacting a cell comprising a DNAvirus with a composition comprising chloroacetoxyquinoline,demethylnobiletin, propanil, aminoethoxydiphenylborane,5-nitro-2-phenylpropylaminobenzoic acid,3beta-hydroxyisoallospirost-9(11)-ene, leoidin, picropodophyllotoxin,thiabendazole, harmane, 6,4′-dihydroxyflavone, gentiopicroside,(R)-angolensin, ptaeroxylin, dipyridamole, nabumetone, rosiglitazone,diltiazem hydrochloride, betamethasone, ichthynone, amcinonide,riluzole, flufenamic acid, chrysin, dictamnine, piplartine, peucenin,methoxyvone, isotretinoin, chloroxylenol, tomatine, primuletin,mefenamic acid, diethylstilbestrol, chloramphenicol palmitate,methylxanthoxylin, 1-alaninol, diclofenac sodium, flunixin, meglumine,dehydroabietamide, pachyrrhizin, dicumarol, diffractic acid, acemetacin,ginkgolic acid, xanthone, fusidic acid, polymyxin b sulfate, pyrantelpamoate, 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one, miconazolenitrate, candesartan cilextil, endosulfan, dioxybenzone, tolfenamicacid, mefloquine, 2-methoxyxanthone, 3-hydroxy-4-(succin-2-yl)-caryolanedelta-lactone, 5,7-dihydroxyflavone, avocadanofuran, benzo(a)pyrene,beta-dihydrogedunol, decahydrogambogic acid, diosmetin, niloticin,pectolinarin, totarol acetate, 8-chloroadenosine, 3-deazaadenosine,O6-cyclohexylmethylguanine, 4-estren-3-beta 17-beta-diol 17-acetate,5-beta-pregnan-3-alpha 6-alpha 20-beta-triol 20-acetate, or4-pregnen-3-beta 20-beta-diol 20-acetate, or any combination thereof.

In some aspects, the invention provides methods of inhibiting viralreplication, the methods comprising contacting a cell comprising a DNAvirus with a composition comprising endosulfan, candesartan cilextil,mefenamic acid, fusidic acid, tolfenamic acid, mefloquine, isotretinon,diclofenac sodium, diltiazem hydrochloride, miconazole nitrate, flunixinmeglumine, propanil, dehydroabietamide, diffractic acid, harmane,xanthone, methoxyvone, or any combination thereof.

In some aspects, the invention provides methods of inhibiting viralreplication, the methods comprising contacting a cell comprising a DNAvirus with a composition comprising mefloquine.

In some aspects, the invention provides methods of inhibiting viralreplication, the methods comprising contacting a cell comprising a DNAvirus with a composition comprising R*,S*-mefloquine.

In some embodiments of the methods described herein, the compositionfurther comprises a pharmaceutically acceptable carrier.

In some embodiments of any of the methods described herein, the DNAvirus is a herpes virus, pox virus, parvovirus, or polyomavirus. In someembodiments, the DNA virus is a polyomavirus. In some embodiments, thepolyomavirus is JC virus. In some embodiments, the polyomavirus is BKvirus.

In some embodiments of the methods described herein, a composition iscontacted to a virus-infected cell in a subject. In some embodiments,the cell is a brain cell, a neuron, a kidney cell, or any other cell ina subject. Accordingly, in some embodiments a composition isadministered to a subject. In some embodiments, the subject is suspectedof having a viral infection (e.g., in at least one cell or tissue type).A subject suspected of having a viral infection may be a subject thathas been identified as having, or known to have, a DNA virus infection(e.g., a JCV or BKV infection). In addition, or alternatively, a subjectsuspected of having a viral infection may be a subject that has beenidentified as being at risk of, or is known to be at risk of, a DNAvirus infection (e.g., a JCV or BKV infection). It should beappreciated, that a subject may be identified as having an infection bydirectly detecting one or more viral molecules (e.g., RNA, DNA, protein,etc., or any combination thereof). However, a subject may be identifiedas having an infection indirectly by detecting one or more indicia of aninfection (e.g., one or more symptoms, one or more serum antibodiesagainst a viral molecule, etc., or any combination thereof). In someembodiments, one or more symptoms of a viral infection may be used toidentify a subject as being at risk of a viral infection. A subject alsomay be identified as being at risk of a viral infection if the subjecthas a reduced or suppressed immune system (e.g., due to a disease,condition, or treatment, or a combination thereof as described in moredetail herein). In some embodiments, the subject has been identified ashaving a JC virus infection in the CNS. In some embodiments, the subjecthas been identified as having a BK virus infection in the kidney. Itshould be appreciated that one or more of the compositions describedherein may be administered to a subject on the basis that the subject issuspected of having a DNA virus infection. For example, a compositionmay be administered to a subject, because the subject had beenidentified as having, or known to have, or identified as being at riskof having, or known to be at risk of having, a DNA virus infection.Accordingly, in some embodiments a subject is evaluated to determinewhether the subject has, or is at risk of having, a DNA virus infection(e.g., a JCV or BKV infection), and a composition described herein isadministered to the subject if they are found to have, or be at risk ofhaving, the DNA virus infection. In some embodiments, a compositiondescribed herein is not administered to a subject that is identified orknown to be virus free and/or risk free.

Accordingly, in some embodiments of the invention a subject is evaluated(e.g., monitored) to determine whether the subject has a sign or symptomof, or is at risk for, a DNA virus infection or proliferation. In someembodiments, this evaluation involves determining whether the subjecthas a symptom of a disease or condition associated with DNA virusactivation (e.g., a symptom of PML). If the subject is identified ashaving a sign or symptom of, or as being at risk for, a DNA virus (e.g.,JCV or BKV) infection or proliferation, the subject is treated with oneor more compounds or compositions of the invention. In some embodiments,a composition comprising chloroacetoxyquinoline, demethylnobiletin,propanil, aminoethoxydiphenylborane, 5-nitro-2-phenylpropylaminobenzoicacid, 3beta-hydroxyisoallospirost-9(11)-ene, leoidin,picropodophyllotoxin, thiabendazole, harmane, 6,4′-dihydroxyflavone,gentiopicroside, (R)-angolensin, ptaeroxylin, dipyridamole, nabumetone,rosiglitazone, diltiazem hydrochloride, betamethasone, ichthynone,amcinonide, riluzole, flufenamic acid, chrysin, dictamnine, piplartine,peucenin, methoxyvone, isotretinoin, chloroxylenol, tomatine,primuletin, mefenamic acid, diethylstilbestrol, chloramphenicolpalmitate, methylxanthoxylin, 1-alaninol, diclofenac sodium, flunixin,meglumine, dehydroabietamide, pachyrrhizin, dicumarol, diffractic acid,acemetacin, ginkgolic acid, xanthone, fusidic acid, polymyxin b sulfate,pyrantel pamoate, 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one,miconazole nitrate, candesartan cilextil, endosulfan, dioxybenzone,tolfenamic acid, mefloquine, 2-methoxyxanthone,3-hydroxy-4-(succin-2-yl)-caryolane delta-lactone, 5,7-dihydroxyflavone,avocadanofuran, benzo(a)pyrene, beta-dihydrogedunol, decahydrogambogicacid, diosmetin, niloticin, pectolinarin, totarol acetate,8-chloroadenosine, 3-deazaadenosine, 06-cyclohexylmethylguanine,4-estren-3-beta 17-beta-diol 17-acetate, 5-beta-pregnan-3-alpha 6-alpha20-beta-triol 20-acetate, or 4-pregnen-3-beta 20-beta-diol 20-acetate,or any combination thereof, is administered to the subject. In someembodiments, a composition comprising candesartan cilextil, mefenamicacid, fusidic acid, tolfenamic acid, mefloquine, isotretinon, diclofenacsodium, diltiazem hydrochloride, miconazole nitrate, flunixin meglumine,propanil, dehydroabietamide, diffractic acid, harmane, xanthone, ormethoxyvone, or any combination thereof, is administered to the subject.

In some embodiments, the subject (e.g., a subject that has beenidentified and treated based on the identification) is evaluated (e.g.,monitored) for one or more signs or symptoms of DNA virus infection orproliferation after receiving a compound or composition of theinvention. For example, in some embodiments of the invention a subjectmay be evaluated to determine or confirm the reduction of at least onesymptom of DNA virus (e.g., JCV or BKV) infection or proliferation. Incertain embodiments, a subject may be evaluated to determine or confirmthat no signs or symptoms of DNA virus (e.g., JCV or BKV) infection orproliferation have developed in the subject. In some embodiments, asubject may be evaluated to determine or confirm that any detectablesigns or symptoms of DNA virus (e.g., JCV or BKV) infection orproliferation have been maintained at a stable level, or that theirdevelopment has been slowed or reversed. In some embodiments, animmunomodulatory treatment or therapy may be altered (e.g., increased,decreased, substituted, or discontinued) based on the evaluation (e.g.,monitoring) of the subject. In some embodiments, a treatment or therapywith one or more compounds or compositions of the invention may bealtered (e.g., increased, decreased, substituted, or discontinued) basedon the evaluation (e.g., monitoring) of the subject before, after, orduring, an immunomodulatory therapy.

In some aspects, the invention provides methods of inhibiting viralreplication in a subject, the methods comprising administering acomposition to a subject suspected of having a DNA virus infection,wherein the composition comprises chloroacetoxyquinoline,demethylnobiletin, propanil, aminoethoxydiphenylborane,5-nitro-2-phenylpropylaminobenzoic acid,3beta-hydroxyisoallospirost-9(11)-ene, leoidin, picropodophyllotoxin,thiabendazole, harmane, 6,4′-dihydroxyflavone, gentiopicroside,(R)-angolensin, ptaeroxylin, dipyridamole, nabumetone, rosiglitazone,diltiazem hydrochloride, betamethasone, ichthynone, amcinonide,riluzole, flufenamic acid, chrysin, dictamnine, piplartine, peucenin,methoxyvone, isotretinoin, chloroxylenol, tomatine, primuletin,mefenamic acid, diethylstilbestrol, chloramphenicol palmitate,methylxanthoxylin, 1-alaninol, diclofenac sodium, flunixin, meglumine,dehydroabietamide, pachyrrhizin, dicumarol, diffractic acid, acemetacin,ginkgolic acid, xanthone, fusidic acid, polymyxin b sulfate, pyrantelpamoate, 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one, miconazolenitrate, candesartan cilextil, endosulfan, dioxybenzone, tolfenamicacid, mefloquine, 2-methoxyxanthone, 3-hydroxy-4-(succin-2-yl)-caryolanedelta-lactone, 5,7-dihydroxyflavone, avocadanofuran, benzo(a)pyrene,beta-dihydrogedunol, decahydrogambogic acid, diosmetin, niloticin,pectolinarin, totarol acetate, 8-chloroadenosine, 3-deazaadenosine,06-cyclohexylmethylguanine, 4-estren-3-beta 17-beta-diol 17-acetate,5-beta-pregnan-3-alpha 6-alpha 20-beta-triol 20-acetate, or4-pregnen-3-beta 20-beta-diol 20-acetate, or any combination thereof, inan amount sufficient to inhibit DNA viral replication in the subject.

In some aspects, the invention provides methods of inhibiting viralreplication in a subject, the methods comprising administering acomposition to a subject suspected of having a DNA virus infection,wherein the composition comprises endosulfan, candesartan cilextil,mefenamic acid, fusidic acid, tolfenamic acid, mefloquine, isotretinon,diclofenac sodium, diltiazem hydrochloride, miconazole nitrate, flunixinmeglumine, propanil, dehydroabietamide, diffractic acid, harmane,xanthone, methoxyvone, or any combination thereof, in an amountsufficient to inhibit DNA viral replication in the subject.

In some embodiments of the methods presented herein, the DNA virusinfection is a herpes virus, pox virus, parvovirus, or polyomavirusinfection. In some embodiments of the methods presented herein the DNAvirus infection is polyomavirus infection. In some embodiments of themethods presented herein the polyomavirus is JC virus. In someembodiments of the methods presented herein the polyomavirus is BKvirus.

In some embodiments of the methods presented herein, the subject hasbeen identified as having a JC virus infection of the CNS. In someembodiments of the methods presented herein, the subject has beenidentified as having a BK virus infection of the kidney.

In some embodiments of the methods presented herein the subject isidentified as being at risk of a DNA virus infection. In someembodiments of the methods presented herein, the subject is undergoing,or has been undergoing, an immunomodulatory treatment. In someembodiments of the methods presented herein, the immunomodulatorytreatment comprises the administration of a VLA-4 antibody. In someembodiments of the methods presented herein, the VLA-4 antibody isnatalizumab.

In some aspects, the invention provides methods of reducing the risk ofa DNA virus infection in a subject, the methods comprising administeringto the subject a composition comprising chloroacetoxyquinoline,demethylnobiletin, propanil, aminoethoxydiphenylborane,5-nitro-2-phenylpropylaminobenzoic acid,3beta-hydroxyisoallospirost-9(11)-ene, leoidin, picropodophyllotoxin,thiabendazole, harmane, 6,4′-dihydroxyflavone, gentiopicroside,(R)-angolensin, ptaeroxylin, dipyridamole, nabumetone, rosiglitazone,diltiazem hydrochloride, betamethasone, ichthynone, amcinonide,riluzole, flufenamic acid, chrysin, dictamnine, piplartine, peucenin,methoxyvone, isotretinoin, chloroxylenol, tomatine, primuletin,mefenamic acid, diethylstilbestrol, chloramphenicol palmitate,methylxanthoxylin, 1-alaninol, diclofenac sodium, flunixin, meglumine,dehydroabietamide, pachyrrhizin, dicumarol, diffractic acid, acemetacin,ginkgolic acid, xanthone, fusidic acid, polymyxin b sulfate, pyrantelpamoate, 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one, miconazolenitrate, candesartan cilextil, endosulfan, dioxybenzone, tolfenamicacid, mefloquine, 2-methoxyxanthone, 3-hydroxy-4-(succin-2-yl)-caryolanedelta-lactone, 5,7-dihydroxyflavone, avocadanofuran, benzo(a)pyrene,beta-dihydrogedunol, decahydrogambogic acid, diosmetin, niloticin,pectolinarin, totarol acetate, 8-chloroadenosine, 3-deazaadenosine,O6-cyclohexylmethylguanine, 4-estren-3-beta 17-beta-diol 17-acetate,5-beta-pregnan-3-alpha 6-alpha 20-beta-triol 20-acetate, or4-pregnen-3-beta 20-beta-diol 20-acetate, or any combination thereof inan amount sufficient to inhibit DNA viral infection.

In some aspects, the invention provides methods of reducing the risk ofa DNA virus infection in a subject, the methods comprising administeringto the subject a composition comprising endosulfan, candesartancilextil, mefenamic acid, fusidic acid, tolfenamic acid, mefloquine,isotretinon, diclofenac sodium, diltiazem hydrochloride, miconazolenitrate, flunixin meglumine, propanil, dehydroabietamide, diffracticacid, harmane, xanthone, methoxyvone or combinations thereof in anamount sufficient to inhibit DNA viral infection.

In some aspects, the invention provides methods of reducing the risk ofPML, the methods comprising administering to a subject having PML, or atrisk of PML, a composition comprising chloroacetoxyquinoline,demethylnobiletin, propanil, aminoethoxydiphenylborane,5-nitro-2-phenylpropylaminobenzoic acid,3beta-hydroxyisoallospirost-9(11)-ene, leoidin, picropodophyllotoxin,thiabendazole, harmane, 6,4′-dihydroxyflavone, gentiopicroside,(R)-angolensin, ptaeroxylin, dipyridamole, nabumetone, rosiglitazone,diltiazem hydrochloride, betamethasone, ichthynone, amcinonide,riluzole, flufenamic acid, chrysin, dictamnine, piplartine, peucenin,methoxyvone, isotretinoin, chloroxylenol, tomatine, primuletin,mefenamic acid, diethylstilbestrol, chloramphenicol palmitate,methylxanthoxylin, 1-alaninol, diclofenac sodium, flunixin, meglumine,dehydroabietamide, pachyrrhizin, dicumarol, diffractic acid, acemetacin,ginkgolic acid, xanthone, fusidic acid, polymyxin b sulfate, pyrantelpamoate, 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one, miconazolenitrate, candesartan cilextil, endosulfan, dioxybenzone, tolfenamicacid, mefloquine, 2-methoxyxanthone, 3-hydroxy-4-(succin-2-yl)-caryolanedelta-lactone, 5,7-dihydroxyflavone, avocadanofuran, benzo(a)pyrene,beta-dihydrogedunol, decahydrogambogic acid, diosmetin, niloticin,pectolinarin, totarol acetate, 8-chloroadenosine, 3-deazaadenosine,O6-cyclohexylmethylguanine, 4-estren-3-beta 17-beta-diol 17-acetate,5-beta-pregnan-3-alpha 6-alpha 20-beta-triol 20-acetate, or4-pregnen-3-beta 20-beta-diol 20-acetate, or any combination thereof inan amount sufficient to inhibit DNA viral infection.

In some aspects, the invention provides methods of reducing the risk ofPML, the methods comprising administering to a subject having PML, or atrisk of PML, a composition comprising endosulfan, candesartan cilextil,mefenamic acid, fusidic acid, tolfenamic acid, mefloquine, isotretinon,diclofenac sodium, diltiazem hydrochloride, miconazole nitrate, flunixinmeglumine, propanil, dehydroabietamide, diffractic acid, harmane,xanthone, methoxyvone or combinations thereof in an amount sufficient toinhibit DNA viral infection.

In some aspects, the invention provides methods of reducing the risk ofPML, the methods comprising administering to a subject having PML, or atrisk of PML, a composition comprising an arylalkanoic acid.

In some embodiments of the methods presented herein, the composition isadministered in a dosage sufficient to reduce the number of JCV infectedcells in an in vitro assay by more than 25%, more than 40%, more than50%, more than 75%, or more than 80%.

In some embodiments of the methods presented herein, the compositioncomprises a compound having an anti-JCV IC₅₀<20 μM and a therapeuticindex IC₅₀/TC₅₀<0.5.

In some embodiments of the methods presented herein, the methods furthercomprise administering an antiviral agent.

In some embodiments of the methods presented herein, the methods furthercomprise performing a plasma exchange.

In some aspects, the invention provides a pharmaceutical compositionand/or a kit comprising natalizumab and one or more compounds selectedfrom the group consisting of chloroacetoxyquinoline, demethylnobiletin,propanil, aminoethoxydiphenylborane, 5-nitro-2-phenylpropylaminobenzoicacid, 3beta-hydroxyisoallospirost-9(11)-ene, leoidin,picropodophyllotoxin, thiabendazole, harmane, 6,4′-dihydroxyflavone,gentiopicroside, (R)-angolensin, ptaeroxylin, dipyridamole, nabumetone,rosiglitazone, diltiazem hydrochloride, betamethasone, ichthynone,amcinonide, riluzole, flufenamic acid, chrysin, dictamnine, piplartine,peucenin, methoxyvone, isotretinoin, chloroxylenol, tomatine,primuletin, mefenamic acid, diethylstilbestrol, chloramphenicolpalmitate, methylxanthoxylin, 1-alaninol, diclofenac sodium, flunixin,meglumine, dehydroabietamide, pachyrrhizin, dicumarol, diffractic acid,acemetacin, ginkgolic acid, xanthone, fusidic acid, polymyxin b sulfate,pyrantel pamoate, 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one,miconazole nitrate, candesartan cilextil, endosulfan, dioxybenzone,tolfenamic acid, mefloquine, 2-methoxyxanthone,3-hydroxy-4-(succin-2-yl)-caryolane delta-lactone, 5,7-dihydroxyflavone,avocadanofuran, benzo(a)pyrene, beta-dihydrogedunol, decahydrogambogicacid, diosmetin, niloticin, pectolinarin, totarol acetate,8-chloroadenosine, 3-deazaadenosine, 06-cyclohexylmethylguanine,4-estren-3-beta 17-beta-diol 17-acetate, 5-beta-pregnan-3-alpha 6-alpha20-beta-triol 20-acetate, or 4-pregnen-3-beta 20-beta-diol 20-acetate,and any combination thereof.

In some aspects, the invention provides a pharmaceutical compositionand/or a kit comprising natalizumab and one or more compounds selectedfrom the group consisting of endosulfan, candesartan cilextil, mefenamicacid, fusidic acid, tolfenamic acid, mefloquine, isotretinon, diclofenacsodium, diltiazem hydrochloride, miconazole nitrate, flunixin meglumine,propanil, dehydroabietamide, diffractic acid, harmane, xanthone,methoxyvone and combinations thereof.

In some embodiments, the invention provides methods of treatmentcomprising administering to a subject having PML, or at risk of PML, atherapeutically effective amount of a pharmaceutical compositioncomprising chloroacetoxyquinoline, demethylnobiletin, propanil,aminoethoxydiphenylborane, 5-nitro-2-phenylpropylaminobenzoic acid,3beta-hydroxyisoallospirost-9(11)-ene, leoidin, picropodophyllotoxin,thiabendazole, harmane, 6,4′-dihydroxyflavone, gentiopicroside,R-angolensin, ptaeroxylin, dipyridamole, nabumetone, rosiglitazone,diltiazem hydrochloride, betamethasone, ichthynone, amcinonide,riluzole, flufenamic acid, chrysin, dictamnine, piplartine, peucenin,methoxyvone, isotretinoin, chloroxylenol, tomatine, primuletin,mefenamic acid, diethylstilbestrol, chloramphenicol palmitate,methylxanthoxylin, 1-alaninol, diclofenac sodium, flunixin, meglumine,dehydroabietamide, pachyrrhizin, dicumarol, diffractic acid, acemetacin,ginkgolic acid, xanthone, fusidic acid, polymyxin b sulfate, pyrantelpamoate, 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one, miconazolenitrate, candesartan cilextil, endosulfan, dioxybenzone, tolfenamicacid, mefloquine([2,8-bis(trifluoromethyl)quinolin-4-yl]-piperidin-2-ylmethanol),2-methoxyxanthone, 3-hydroxy-4-(succin-2-yl)-caryolane delta-lactone,5,7-dihydroxyflavone, avocadanofuran, benzo(a)pyrene,beta-dihydrogedunol, decahydrogambogic acid, diosmetin, niloticin,pectolinarin, totarol acetate, 8-chloroadenosine, 3-deazaadenosine,O6-cyclohexylmethylguanine, roscovitine, 4-estren-3-beta 17-beta-diol17-acetate, 5-beta-pregnan-3-alpha 6-alpha 20-beta-triol 20-acetate,4-pregnen-3-beta 20-beta-diol 20-acetate, or any combination of two ormore thereof.

In some embodiments, the invention provides methods of treatmentcomprising administering to a subject having PML, or at risk of PML, atherapeutically effective amount of a pharmaceutical compositioncomprising endosulfan, candesartan cilextil, mefenamic acid, fusidicacid, tolfenamic acid, mefloquine, isotretinon, diclofenac sodium,diltiazem hydrochloride, miconazole nitrate, flunixin meglumine,propanil, dehydroabietamide, diffractic acid, harmane, xanthone,methoxyvone or combinations thereof.

In some embodiments, the invention provides methods of treatmentcomprising administering to a subject having PML, or at risk of PML, atherapeutically effective amount of a pharmaceutical compositioncomprising mefloquine.

In some embodiments, the invention provides methods of treatmentcomprising administering to a subject having PML, or at risk of PML, atherapeutically effective amount of a pharmaceutical compositioncomprising R,S-mefloquine.

In some embodiments, the invention provides methods of treatmentcomprising administering to a subject having PML, or at risk of PML, atherapeutically effective amount of a pharmaceutical compositioncomprising a compound with the same mode of action as endosulfan,candesartan cilextil, mefenamic acid, fusidic acid, tolfenamic acid,mefloquine, isotretinon, diclofenac sodium, diltiazem hydrochloride,miconazole nitrate, flunixin meglumine, propanil, dehydroabietamide,diffractic acid, harmane, xanthone, methoxyvone or any combinationthereof.

In some embodiments, the invention provides methods of treatmentcomprising administering to a subject having PML, or at risk of PML, atherapeutically effective amount of a pharmaceutical compositioncomprising a compound comprising an adenosine, guanine, estren, orpregnan component.

In some embodiments, the invention provides methods of treatmentcomprising administering to a subject having PML, or at risk of PML, atherapeutically effective amount of a pharmaceutical compositioncomprising a compound comprising an arylalkanoic acid.

In some embodiments, the invention provides methods of treating asubject infected with a DNA virus, or suspected of being infected with aDNA virus, the method comprising administering to a subject atherapeutically effective amount of a pharmaceutical compositioncomprising chloroacetoxyquinoline, demethylnobiletin, propanil,aminoethoxydiphenylborane, 5-nitro-2-phenylpropylaminobenzoic acid,3beta-hydroxyisoallospirost-9(11)-ene, leoidin, picropodophyllotoxin,thiabendazole, harmane, 6,4′-dihydroxyflavone, gentiopicroside,R-angolensin, ptaeroxylin, dipyridamole, nabumetone, rosiglitazone,diltiazem hydrochloride, betamethasone, ichthynone, amcinonide,riluzole, flufenamic acid, chrysin, dictamnine, piplartine, peucenin,methoxyvone, isotretinoin, chloroxylenol, tomatine, primuletin,mefenamic acid, diethylstilbestrol, chloramphenicol palmitate,methylxanthoxylin, 1-alaninol, diclofenac sodium, flunixin, meglumine,dehydroabietamide, pachyrrhizin, dicumarol, diffractic acid, acemetacin,ginkgolic acid, xanthone, fusidic acid, polymyxin b sulfate, pyrantelpamoate, 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one, miconazolenitrate, candesartan cilextil, endosulfan, dioxybenzone, tolfenamicacid, mefloquine([2,8-bis(trifluoromethyl)quinolin-4-yl]-piperidin-2-ylmethanol),2-methoxyxanthone, 3-hydroxy-4-(succin-2-yl)-caryolane delta-lactone,5,7-dihydroxyflavone, avocadanofuran, benzo(a)pyrene,beta-dihydrogedunol, decahydrogambogic acid, diosmetin, niloticin,pectolinarin, totarol acetate, 8-chloroadenosine, 3-deazaadenosine,O6-cyclohexylmethylguanine, roscovitine, 4-estren-3-beta 17-beta-diol17-acetate, 5-beta-pregnan-3-alpha 6-alpha 20-beta-triol 20-acetate,4-pregnen-3-beta 20-beta-diol 20-acetate, or any combination of two ormore thereof.

In some embodiments, the invention provides methods of treatment for asubject infected with a DNA virus, or suspected of being infected with aDNA virus, wherein the DNA virus is selected from the group consistingof herpes virus, pox virus, parvovirus and polyomavirus. In someembodiments, the invention provides methods of treatment for a subjectinfected with JC virus, or suspected of being infected with JC virus. Insome embodiments, the invention provides methods of treatment for asubject infected with BK virus, or suspected of being infected with BKvirus. In some embodiments, the invention provides methods of treatmentfor a subject infected with a JC virus, wherein JC virus infection ischaracterized by the presence of JC virus in the central nervous system(CNS). In some embodiments, the invention provides methods of treatmentfor a subject infected with a BK virus, wherein BK virus infection ischaracterized by the presence of BK virus in the kidney.

In some embodiments, the invention provides methods of preventing orsuppressing PML, the method comprising administering to a subject atherapeutically effective amount of a pharmaceutical compositioncomprising chloroacetoxyquinoline, demethylnobiletin, propanil,aminoethoxydiphenylborane, 5-nitro-2-phenylpropylaminobenzoic acid,3beta-hydroxyisoallospirost-9(11)-ene, leoidin, picropodophyllotoxin,thiabendazole, harmane, 6,4′-dihydroxyflavone, gentiopicroside,R-angolensin, ptaeroxylin, dipyridamole, nabumetone, rosiglitazone,diltiazem hydrochloride, betamethasone, ichthynone, amcinonide,riluzole, flufenamic acid, chrysin, dictamnine, piplartine, peucenin,methoxyvone, isotretinoin, chloroxylenol, tomatine, primuletin,mefenamic acid, diethylstilbestrol, chloramphenicol palmitate,methylxanthoxylin, 1-alaninol, diclofenac sodium, flunixin, meglumine,dehydroabietamide, pachyrrhizin, dicumarol, diffractic acid, acemetacin,ginkgolic acid, xanthone, fusidic acid, polymyxin b sulfate, pyrantelpamoate, 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one, miconazolenitrate, candesartan cilextil, endosulfan, dioxybenzone, tolfenamicacid, mefloquine([2,8-bis(trifluoromethyl)quinolin-4-yl]-piperidin-2-ylmethanol),2-methoxyxanthone, 3-hydroxy-4-(succin-2-yl)-caryolane delta-lactone,5,7-dihydroxyflavone, avocadanofuran, benzo(a)pyrene,beta-dihydrogedunol, decahydrogambogic acid, diosmetin, niloticin,pectolinarin, totarol acetate, 8-chloroadenosine, 3-deazaadenosine,O6-cyclohexylmethylguanine, roscovitine, 4-estren-3-beta 17-beta-diol17-acetate, 5-beta-pregnan-3-alpha 6-alpha 20-beta-triol 20-acetate,4-pregnen-3-beta 20-beta-diol 20-acetate, or any combination of two ormore thereof.

In some embodiments, the invention provides methods of treatment of asubject having PML, or suspected of having PML, the method comprisingadministering to a subject a therapeutically effective amount of apharmaceutical composition comprising a compound in a dosage sufficientto reduce the number of JCV infected cells in an in vitro assay by morethan 40%.

In some embodiments, the invention provides methods of treatment of asubject having PML, or suspected of having PML, comprising administeringto a subject a therapeutically effective amount of a pharmaceuticalcomposition comprising a compound, wherein the compound has an anti-JCVIC₅₀<20 μM and a therapeutic index IC₅₀/TC₅₀<0.5.

In some embodiments, the invention provides methods of treatment for asubject infected with a DNA virus, or suspected of being infected with aDNA virus, or of a subject having PML, or suspected of having PML,wherein the subject is undergoing, or has been undergoing,immunomodulatory treatment.

In some embodiments, the invention provides methods of preventing viralinfection in a person undergoing immunomodulatory treatment, the methodcomprising administering to a subject a therapeutically effective amountof a pharmaceutical composition comprising chloroacetoxyquinoline,demethylnobiletin, propanil, aminoethoxydiphenylborane,5-nitro-2-phenylpropylaminobenzoic acid,3beta-hydroxyisoallospirost-9(11)-ene, leoidin, picropodophyllotoxin,thiabendazole, harmane, 6,4′-dihydroxyflavone, gentiopicroside,R-angolensin, ptaeroxylin, dipyridamole, nabumetone, rosiglitazone,diltiazem hydrochloride, betamethasone, ichthynone, amcinonide,riluzole, flufenamic acid, chrysin, dictamnine, piplartine, peucenin,methoxyvone, isotretinoin, chloroxylenol, tomatine, primuletin,mefenamic acid, diethylstilbestrol, chloramphenicol palmitate,methylxanthoxylin, 1-alaninol, diclofenac sodium, flunixin, meglumine,dehydroabietamide, pachyrrhizin, dicumarol, diffractic acid, acemetacin,ginkgolic acid, xanthone, fusidic acid, polymyxin b sulfate, pyrantelpamoate, 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one, miconazolenitrate, candesartan cilextil, endosulfan, dioxybenzone, tolfenamicacid, mefloquine([2,8-bis(trifluoromethyl)quinolin-4-yl]-piperidin-2-ylmethanol),2-methoxyxanthone, 3-hydroxy-4-(succin-2-yl)-caryolane delta-lactone,5,7-dihydroxyflavone, avocadanofuran, benzo(a)pyrene,beta-dihydrogedunol, decahydrogambogic acid, diosmetin, niloticin,pectolinarin, totarol acetate, 8-chloroadenosine, 3-deazaadenosine,06-cyclohexylmethylguanine, roscovitine, 4-estren-3-beta 17-beta-diol17-acetate, 5-beta-pregnan-3-alpha 6-alpha 20-beta-triol 20-acetate,4-pregnen-3-beta 20-beta-diol 20-acetate, or any combination of two ormore thereof.

In some embodiments, the immunomodulatory treatment comprises theadministration of a VLA-4 antibody. In some embodiments, theimmunomodulatory treatment comprises the administration of natalizumab.

In some embodiments, the invention provides methods of treatment for asubject infected with a DNA virus, or suspected of being infected with aDNA virus, or of a subject having PML, or suspected of having PMLcomprising administering one or more compounds of Table 1 and anantiviral therapeutic.

In some embodiments, the invention provides methods of treatment for asubject infected with a DNA virus, or suspected of being infected with aDNA virus, or of a subject having PML, or suspected of having PML,comprising administering one or more compounds of Table 1 and anadjuvant.

In some embodiments, the invention provides methods of treatment for asubject infected with a DNA virus, or suspected of being infected with aDNA virus, or of a subject having PML, or suspected of having PML,comprising administering multiple compounds selected from the groupconsisting of chloroacetoxyquinoline, demethylnobiletin, propanil,aminoethoxydiphenylborane, 5-nitro-2-phenylpropylaminobenzoic acid,3beta-hydroxyisoallospirost-9(11)-ene, leoidin, picropodophyllotoxin,thiabendazole, harmane, 6,4′-dihydroxyflavone, gentiopicroside,R-angolensin, ptaeroxylin, dipyridamole, nabumetone, rosiglitazone,diltiazem hydrochloride, betamethasone, ichthynone, amcinonide,riluzole, flufenamic acid, chrysin, dictamnine, piplartine, peucenin,methoxyvone, isotretinoin, chloroxylenol, tomatine, primuletin,mefenamic acid, diethylstilbestrol, chloramphenicol palmitate,methylxanthoxylin, 1-alaninol, diclofenac sodium, flunixin, meglumine,dehydroabietamide, pachyrrhizin, dicumarol, diffractic acid, acemetacin,ginkgolic acid, xanthone, fusidic acid, polymyxin b sulfate, pyrantelpamoate, 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one, miconazolenitrate, candesartan cilextil, endosulfan, dioxybenzone, tolfenamicacid, mefloquine([2,8-bis(trifluoromethyl)quinolin-4-yl]-piperidin-2-ylmethanol),2-methoxyxanthone, 3-hydroxy-4-(succin-2-yl)-caryolane delta-lactone,5,7-dihydroxyflavone, avocadanofuran, benzo(a)pyrene,beta-dihydrogedunol, decahydrogambogic acid, diosmetin, niloticin,pectolinarin, totarol acetate, 8-chloroadenosine, 3-deazaadenosine,O6-cyclohexylmethylguanine, roscovitine, 4-estren-3-beta 17-beta-diol17-acetate, 5-beta-pregnan-3-alpha 6-alpha 20-beta-triol 20-acetate, and4-pregnen-3-beta 20-beta-diol 20-acetate

In some embodiments, the invention provides methods of treatment for asubject infected with a DNA virus, or suspected of being infected with aDNA virus, or of a subject having PML, or suspected of having PML,comprising administering one or more compounds of Table 1 and performinga plasma exchange.

In some embodiments, the invention provides a pharmaceutical compositioncomprising natalizumab and one or more compounds selected from the groupconsisting of chloroacetoxyquinoline, demethylnobiletin, propanil,aminoethoxydiphenylborane, 5-nitro-2-phenylpropylaminobenzoic acid,3beta-hydroxyisoallospirost-9(11)-ene, leoidin, picropodophyllotoxin,thiabendazole, harmane, 6,4′-dihydroxyflavone, gentiopicroside,R-angolensin, ptaeroxylin, dipyridamole, nabumetone, rosiglitazone,diltiazem hydrochloride, betamethasone, ichthynone, amcinonide,riluzole, flufenamic acid, chrysin, dictamnine, piplartine, peucenin,methoxyvone, isotretinoin, chloroxylenol, tomatine, primuletin,mefenamic acid, diethylstilbestrol, chloramphenicol palmitate,methylxanthoxylin, 1-alaninol, diclofenac sodium, flunixin, meglumine,dehydroabietamide, pachyrrhizin, dicumarol, diffractic acid, acemetacin,ginkgolic acid, xanthone, fusidic acid, polymyxin b sulfate, pyrantelpamoate, 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one, miconazolenitrate, candesartan cilextil, endosulfan, dioxybenzone, tolfenamicacid, mefloquine([2,8-bis(trifluoromethyl)quinolin-4-yl]-piperidin-2-ylmethanol),2-methoxyxanthone, 3-hydroxy-4-(succin-2-yl)-caryolane delta-lactone,5,7-dihydroxyflavone, avocadanofuran, benzo(a)pyrene,beta-dihydrogedunol, decahydrogambogic acid, diosmetin, niloticin,pectolinarin, totarol acetate, 8-chloroadenosine, 3-deazaadenosine,06-cyclohexylmethylguanine, roscovitine, 4-estren-3-beta 17-beta-diol17-acetate, 5-beta-pregnan-3-alpha 6-alpha 20-beta-triol 20-acetate and4-pregnen-3-beta 20-beta-diol 20-acetate.

In some embodiments, the invention provides a kit comprising natalizumaband one or more compounds selected from the group consisting ofchloroacetoxyquinoline, demethylnobiletin, propanil,aminoethoxydiphenylborane, 5-nitro-2-phenylpropylaminobenzoic acid,3beta-hydroxyisoallospirost-9(11)-ene, leoidin, picropodophyllotoxin,thiabendazole, harmane, 6,4′-dihydroxyflavone, gentiopicroside,R-angolensin, ptaeroxylin, dipyridamole, nabumetone, rosiglitazone,diltiazem hydrochloride, betamethasone, ichthynone, amcinonide,riluzole, flufenamic acid, chrysin, dictamnine, piplartine, peucenin,methoxyvone, isotretinoin, chloroxylenol, tomatine, primuletin,mefenamic acid, diethylstilbestrol, chloramphenicol palmitate,methylxanthoxylin, 1-alaninol, diclofenac sodium, flunixin, meglumine,dehydroabietamide, pachyrrhizin, dicumarol, diffractic acid, acemetacin,ginkgolic acid, xanthone, fusidic acid, polymyxin b sulfate, pyrantelpamoate, 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one, miconazolenitrate, candesartan cilextil, endosulfan, dioxybenzone, tolfenamicacid, mefloquine([2,8-bis(trifluoromethyl)quinolin-4-yl]-piperidin-2-ylmethanol),2-methoxyxanthone, 3-hydroxy-4-(succin-2-yl)-caryolane delta-lactone,5,7-dihydroxyflavone, avocadanofuran, benzo(a)pyrene,beta-dihydrogedunol, decahydrogambogic acid, diosmetin, niloticin,pectolinarin, totarol acetate, 8-chloroadenosine, 3-deazaadenosine,06-cyclohexylmethylguanine, roscovitine, 4-estren-3-beta 17-beta-diol17-acetate, 5-beta-pregnan-3-alpha 6-alpha 20-beta-triol 20-acetate, and4-pregnen-3-beta 20-beta-diol 20-acetate, and instructions foradministering the compounds.

In some embodiments, the invention provides a method of inhibiting(e.g., reducing or suppressing) replication of a DNA virus in a cellcomprising contacting a cell comprising a DNA virus with a compound,wherein contacting the cell with the compound results in the reductionor suppression of replication of the DNA virus, wherein the compound isselected from the group consisting of chloroacetoxyquinoline,demethylnobiletin, propanil, aminoethoxydiphenylborane,5-nitro-2-phenylpropylaminobenzoic acid,3beta-hydroxyisoallospirost-9(11)-ene, leoidin, picropodophyllotoxin,thiabendazole, harmane, 6,4′-dihydroxyflavone, gentiopicroside,R-angolensin, ptaeroxylin, dipyridamole, nabumetone, rosiglitazone,diltiazem hydrochloride, betamethasone, ichthynone, amcinonide,riluzole, flufenamic acid, chrysin, dictamnine, piplartine, peucenin,methoxyvone, isotretinoin, chloroxylenol, tomatine, primuletin,mefenamic acid, diethylstilbestrol, chloramphenicol palmitate,methylxanthoxylin, 1-alaninol, diclofenac sodium, flunixin, meglumine,dehydroabietamide, pachyrrhizin, dicumarol, diffractic acid, acemetacin,ginkgolic acid, xanthone, fusidic acid, polymyxin b sulfate, pyrantelpamoate, 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one, miconazolenitrate, candesartan cilextil, endosulfan, dioxybenzone, tolfenamicacid, mefloquine([2,8-bis(trifluoromethyl)quinolin-4-yl]-piperidin-2-ylmethanol),2-methoxyxanthone, 3-hydroxy-4-(succin-2-yl)-caryolane delta-lactone,5,7-dihydroxyflavone, avocadanofuran, benzo(a)pyrene,beta-dihydrogedunol, decahydrogambogic acid, diosmetin, niloticin,pectolinarin, totarol acetate, 8-chloroadenosine, 3-deazaadenosine,O6-cyclohexylmethylguanine, roscovitine, 4-estren-3-beta 17-beta-diol17-acetate, 5-beta-pregnan-3-alpha 6-alpha 20-beta-triol 20-acetate,4-pregnen-3-beta 20-beta-diol 20-acetate and any combination of two ormore thereof.

In some embodiments, the invention provides a method of suppressingreplication of a DNA virus in a cell, wherein the DNA virus is selectedfrom the group consisting of herpes virus, pox virus, parvovirus, JCvirus, and BK virus.

These and other aspects of the invention are described in more detailherein and illustrated by the following non-limiting figures andexamples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a non-limiting embodiment of a decrease in the amountof inhibition with an increase in the amount of neutralizing JCVantibody added;

FIG. 2 illustrates a non-limiting embodiment of the correlation betweeninfection rate and JCV virus concentration;

FIG. 3 illustrates a non-limiting embodiment of detection andmeasurement of cellular infection with JCV, panel (A) shows cellsinfected with JCV, panel (B) shows the number of infected cells plottedagainst the dilution factor of the viral stock used to infect the cells,panels (C) and (D) illustrate the inhibition of JCV in the presence ofvarious dilutions of JCV neutralizing antiserum or cidofovir;

FIG. 4 illustrates a non-limiting embodiment of a flow chart forcompound screening;

FIG. 5 illustrates a non-limiting embodiment of the efficacy ofmefloquine against two different viral strains, panel (A) illustratesSVG-A cells infected with Mad1/SVEΔ JCV virus, panel (B) illustratesprimary human fetal astrocytes infected with Mad1/SVEΔ JCV, and panel(C) illustrates SVG-A cells infected with JCV strain Mad-4;

FIG. 6 illustrates a non-limiting embodiment of the effect of mefloquineon JCV DNA replication;

FIG. 7 illustrates a non-limiting embodiment of the efficacy ofmefloquine against JCV infection;

FIG. 8 illustrates non-limiting embodiments of the efficacy of differentisomers of mefloquine;

FIG. 9 illustrates non-limiting embodiments of the efficacy of differentisomers of mefloquine;

FIG. 10 shows that mefloquine anti-JCV activity is not inhibited bycerebrospinal fluid (CSF);

FIG. 11 illustrates non-limiting embodiments of the dose response ofdrugs with anti-JCV activity;

FIG. 12 illustrates non-limiting embodiments of arylalkanoic acid NSAIDsand their anti-JCV activity;

FIG. 13 illustrates non-limiting embodiments of arylalkanoic acid NSAIDsand their anti-JCV activity;

FIG. 14 illustrates non-limiting embodiments of modeling studies withmefloquine and related compounds;

FIG. 15 illustrates non-limiting embodiments of structures ofJCV-inhibitor compounds; and,

FIG. 16 illustrates non-limiting embodiments of structures ofJCV-inhibitor compounds.

DETAILED DESCRIPTION OF THE INVENTION

Aspects of the invention relate to compositions identified as havinganti-viral activity and their use to prevent or treat viral infection insubjects. In particular, aspects of the invention relate to compositionsfound to inhibit DNA viral activity. In some embodiments, one or morecompositions that inhibit JCV activity are provided and may beadministered to subjects infected or at risk of infection with JCVand/or to subjects with PML, or at risk of developing PML.

Aspects of the invention are based, at least in part, on the surprisingdiscovery that the compounds described in Table 1 reduce the percentageor number of JCV infected cells in a cell-based assay. The compoundsdescribed in Table 1 were not previously known to have anti-JCV activityor any anti-viral activity. Accordingly, one or more compounds describedin Table 1 may be used as described herein.

TABLE 1 Therapeutic Name Reference Indication CAS numberCHLOROACETOXYQUINOLINE antifungal 10173-02-1 DEMETHYLNOBILETIN BiochemBiophys Res Commun. 2005 2174-59-6 Dec 2; 337(4):1330-6. Epub 2005 Oct.10. PROPANIL U.S. Pat. No. 6,060,432 herbicide 709-98-8AMINOETHOXYDIPHENYLBORANE J Neurophysiol. 2005 November; 94(5): 3069-80.Ca release 524-95-8 Epub 2005 Jul. 13. inhibitor, angiogensin IIinhibitor 5-NITRO-2- J Cell Physiol. 1995 January; 162(1): 15-25.chloride channel PHENYLPROPYLAMINOBENZOIC blocker ACID [NPPB]3beta-HYDROXYISOALLOSPIROST- J Nat Prod. 2007 July; 70(7): 1203-6.9(11)-ENE Epub 2007 Jul. 13. LEOIDIN 105350-54-7 PICROPODOPHYLLOTOXINBiol Pharm Bull. 2007 July; 30(7): 1340-3. antineoplastic; 477-47-4 10%cytotoxicity of podophyllotoxin THIABENDAZOLE U.S. Pat. No. 5,840,324anthelmintic 148-79-8 HARMANE intercalating agent, 486-84-0 sedative6,4′-DIHYDROXYFLAVONE antihaemorrhagic 79786-40-6 GENTIOPICROSIDE YaoXue Xue Bao. 2007 May; 42(5): 566-70. antimalarial, larvicide(R)-ANGOLENSIN 4842-48-2 PTAEROXYLIN Biochem Syst Ecol. 2000 Aug. 1;28(7): 713-716. 14729-11-4 DIPYRIDAMOLE U.S. Pat. No. 7,253,155 coronary58-32-2 vasodilator NABUMETONE U.S. Pat. No. 6,544,556 antiinflammatory42924-53-8 ROSIGLITAZONE U.S. Pat. No. 6,673,815 antidiabetic122320-73-4 DILTIAZEM HYDROCHLORIDE U.S. Pat. No. 5,578,321 Ca channel33286-22-5 blocker, coronary vasodilator BETAMETHASONE U.S. Pat. No.6,878,518 glucocorticoid, 378-44-9 antiinflammatory ICHTHYNONE VoprPitan. 1995; (4): 13-6. piscicide 24340-62-3 AMCINONIDE U.S. Pat. No.6,426,339 glucocorticoid, 51022-69-6 antiinflammatory RILUZOLE U.S. Pat.No. 6,660,757 anticonvulsant, 1744-22-5 glutamate release inhibitorFLUFENAMIC ACID U.S. Pat. No. 5,968,551 antiinflammatory, 530-78-9analgesic CHRYSIN U.S. Pat. No. 6,607,755 diuretic 480-40-0 DICTAMNINEPlanta Med. 2006 August; 72(10): 941-3. 484-29-7 PIPLARTINEPhytomedicine. 2007 September; 14(9): 605-12. anti-asthma, 20069-09-4Epub 2007 Mar. 30. antibronchitis PEUCENIN Chem Pharm Bull (Tokyo). 2006January; 54(1): 44-7. 578-72-3 METHOXYVONE Bioorg Med Chem. 2007 Sep.15; 15(18): 6089-95. anabolic Epub 2007 Jun. 26. ISOTRETINON U.S. Pat.No. 6,936,267 antiacne, 4759-48-2 antineoplastic CHLOROXYLENOL U.S. Pat.No. 4,902,501 antibacterial, 88-04-0 topical and urinary TOMATINE U.S.Pat. No. 6,673,357 antifungal, antibacterial, antiinflammatory agentPRIMULETIN 491-78-1 MEFENAMIC ACID U.S. Pat. No. 6,645,520antiinflammatory, 61-68-7 analgesic DIETHYLSTILBESTROL U.S. Pat. No.6,040,306 estrogen 56-53-1 CHLORAMPHENICOL PALMITATE antibacterial,530-43-8 tetratogen METHYLXANTHOXYLIN Phytother Res. 2004 July; 18(7):542-5. 23121-32-6 L-ALANINOL Microbiology. 2005 July; 151(Pt 7):2385-92. antiproliferative 2749-11-3 DICLOFENAC SODIUM U.S. Pat. No.6,387,410 antiinflammatory 15307-79-6 FLUNIXIN MEGLUMINE U.S. Pat. No.6,924,273 analgesic, 42461-84-7 antiinflammatory DEHYDROABIETAMIDE U.S.Pat. No. 4,755,523 PACHYRRHIZIN Trans R Soc Trop Med Hyg. 2004 August;98(8): 451-5. insecticide 10091-01-7 DICUMAROL U.S. Pat. No. 5,024,998anticoagulant 66-76-2 DIFFRACTIC ACID Inflamm Res. 2007 April; 56 Suppl1: 521-2. ACEMETACIN U.S. Pat. No. 7,109,176 antiinflammatory 53164-05-9GINKGOLIC ACID antibacterial, 22910-60-7 antitubercular XANTHONE U.S.Pat. No. 6,927,234 90-47-1 FUSIDIC ACID U.S. Pat. No. 6,462,182antibacterial 6990-06-3 POLYMYXIN B SULFATE U.S. Pat. No. 5,648,397antibacterial 1405-20-5 PYRANTEL PAMOATE U.S. Pat. No. 7,144,878anthelmintic 22204-24-6 4-(3-BUTOXY-4- Eur J Pharmacol. 2003 Mar. 28;465(1-2): 133-9. cAMP PDE METHOXYBENZYL)IMIDAZOLIDIN- inhibitor,inhibits 2-ONE cellular adhesion and superoxide & platelet aggregationMICONAZOLE NITRATE U.S. Pat. No. 6,001,864 antifungal (topical)22832-87-7, 22916-47-8 [miconazole] CANDESARTAN CILEXTIL angiotensin 1170791-09-0 receptor antagonist ENDOSULFAN U.S. Pat. No. 6,294,570insecticide 115-29-7, 959-98-8 (alpha), 33213-65-9 (beta) DIOXYBENZONEU.S. Pat. No. 5,916,544 ultraviolet screen 131-53-3 TOLFENAMIC ACID U.S.Pat. No. 6,685,928 antiinflammatory, 13710-19-5 analgesia MEFLOQUINEU.S. Pat. No. 5,834,505 antimalarial 53230-10-7 2-METHOXYXANTHONEundetermined 1214-20-6 activity 3-HYDROXY-4-(SUCCIN-2-YL)- undeterminedCARYOLANE delta-LACTONE activity 5,7-DIHYDROXYFLAVONE undetermined480-40-0 activity AVOCADANOFURAN undetermined activity BENZO[a]PYRENEcarcinogen, binds to DNA experimental 192-97-2 beta-DIHYDROGEDUNOLundetermined activity DECAHYDROGAMBOGIC ACID undetermined activityDIOSMETIN undetermined 520-34-3 activity NILOTICIN undetermined activityPECTOLINARIN undetermined 28978-02-1 activity TOTAROL ACETATEundetermined activity 8-CHLOROADENOSINE 34408-14-5 3-DEAZAADENOSINE6736-58-9 O6- CYCLOHEXYLMETHYLGUANINE ROSCOVITINE, (S)-ISOMER4-ESTREN-3-BETA, 17-BETA-DIOL 17-ACETATE 5-BETA-PREGNAN-3-ALPHA, 6-ALPHA, 20-BETA-TRIOL 20- ACETATE 4-PREGNEN-3-BETA, 20-BETA- DIOL20-ACETATE

In some embodiments, one or more of the compounds of Table 1 may be used(e.g., combinations of two or more, for example, 2, 3, 4, 5, 6, 7, 8, 9,10, etc.). In some embodiments, a composition comprises multiplecompounds of Table 1. In some embodiments, the treatment or preventionregimen comprises the use of multiple compositions each comprising oneor more compounds of Table 1.

In some embodiments, one or more compounds related to those listed inTable 1 may be used as described herein. For example, structurallyrelated compounds, compounds with a similar mode of action, and/orcompounds that interact with one or more of the same targets (e.g.,viral or cellular targets, such as receptors, intracellular pathwaycomponents or other targets) may be used to treat or prevent DNA virus(e.g., JCV) infection and or activity.

In some embodiments, the invention relates to the use of mefloquine, andcompounds with a similar mode of action as mefloquine, to prevent ortreat DNA viral infection or proliferation (e.g., to reduce the risk ofDNA viral infection or proliferation). Mefloquine is a quinine relatedanti-malarial drug. Non-limiting examples of drugs with a similar modeof action are quinine, chloroquine and halofantrine. Quinines arelysosomotropic drugs that selectively accumulate inside lysosomes. Theuncharged compound rapidly diffuses through the plasma and lysosomalmembranes, while once charged the compound becomes trapped inside theacidic lysosomal compartment of the parasite. In some embodiments, theinvention relates to the treatment or prevention of PML usinglysosomotropic drugs.

In some embodiments, the invention relates to the use of(+)-(R,S)-mefloquine to prevent or treat DNA viral infection orproliferation. In some embodiments, the invention relates to the use of(−)-(S,R)-mefloquine to prevent or treat DNA viral infection orproliferation. In some embodiments, the invention relates to the use ofa racemic mixture of (+)-(R,S)-mefloquine and (−)-(S,R)-mefloquine toprevent or treat DNA viral infection or proliferation. In someembodiments, the invention relates to the use of (R,R)-mefloquine toprevent or treat DNA viral infection or proliferation. In someembodiments, the invention relates to the use of (S,S)-mefloquine toprevent or treat DNA viral infection or proliferation. In someembodiments, the invention relates to the use of a mixture of(R,R)-mefloquine and (S,S)-mefloquine to prevent or treat DNA viralinfection or proliferation. In some embodiments, the invention relatesto the use of one or more of the following mefloquine compounds:(+)-(R,S)-mefloquine, (−)-(S,R)-mefloquine, (R,R)-mefloquine and(S,S)-mefloquine to prevent or treat DNA viral infection orproliferation.

In some embodiments, the invention relates to the use of compounds thatare structurally related to mefloquine to prevent or treat DNA viralinfection or proliferation. In some embodiments, compounds that arestructurally related to mefloquine are compounds that comprise the2,8-bis-trifluoromethyl-quonolin-4-yl structural group. In someembodiments, compounds that are structurally related to mefloquine areare quinolines substituted on either or both rings. In some embodiments,compounds that are structurally related to mefloquine are substitutedindoles. In some embodiments, compounds that are structurally related tomefloquine are substituted 3-indoleacetic acid derivatives. In someembodiments, compounds that are structurally related to mefloquine aresubstituted 6-amino purine derivatives. In some embodiments, compoundsthat are structurally related to mefloquine are substituted 2,6-diaminopurine derivatives. In some embodiments, compounds that are structurallyrelated to mefloquine are substituted imidazopyridin-4-amines. In someembodiments, compounds that are structurally related to mefloquine aresubstituted imidazopyridines. In some embodiments, compounds that arestructurally related to mefloquine are compounds that have a similarshape as mefloquine. In some embodiments, compounds that arestructurally related to mefloquine are 3-deazaadenosine, indomethacin,mefenamic acid, 8-chloroadenosine and O6-cyclohexylmethylguanine (SeeFIGS. 13 and 14). In some embodiments, a compound that is structurallyrelated to mefloquine is roscovitine. In some embodiments, a compoundthat is structurally related to mefloquine is S-roscovitine. In someembodiments, a compound that is structurally related to mefloquine isR-roscovitine. The antiviral effects of R-roscovitine have beendescribed, for instance in Orba et al. (Virology 2008 Jan. 5;370(1):173-83).

In some embodiments, the invention relates to the use of tolfenamicacid, and compounds with the same mode of action as tolfenamic acid, toprevent or treat DNA viral infection or proliferation (e.g., to reducethe risk of DNA viral infection or proliferation). Tolfenamic acid is adrug that belongs to the class of NSAIDs (Non-SteroidalAnti-Inflammatory Drugs), and acts by inhibiting isoforms ofcyclo-oxygenase 1 and 2 (COX 1 and COX2). In some embodiments, theinvention relates to the treatment or prevention of PML using NSAIDs. Insome embodiments, the invention relates to the treatment or preventionof PML using NSAID arylalkanoic acids. In some embodiments, theinvention relates to the treatment or prevention of PML usingarylalkanoic acids.

In some embodiments, the invention relates to the use of compounds thatare structurally related to tolfenamic acid to prevent or treat DNAviral infection or proliferation. In some embodiments, compounds thatare structurally related to tolfenamic acid are compounds that comprisetwo aromatic rings linked by a nitrogen. In some embodiments, compoundsthat are structurally related to tolfenamic acid are arylalkanoic acids.In some embodiments, arylalkanoic acids are secondary amines in whichtwo substituents are substituted and/or unsubstituted aryl groups.Non-limiting examples of arylalkanoic acids are dicolfenac, mefenamicacid, flufenamic acid and flunexin (See FIGS. 12 and 13).

In some embodiments, the invention relates to the use of dioxybenzone(2,2′-Dihydroxy-4-methoxybenzophenone), and compounds with the same modeof action as dioxybenzone, to prevent or treat DNA viral infection orproliferation (e.g., to reduce the risk of DNA viral infection orproliferation). In some embodiments, the invention relates to thetreatment or prevention of PML using compounds that are structurallyrelated to dioxybenzone.

In some embodiments, the invention relates to the use of endosulfan, andcompounds with the same mode of action as endosulfan, to prevent ortreat DNA viral infection or proliferation (e.g., to reduce the risk ofDNA viral infection or proliferation). Endosulfan can act as a proteinchannel agonist. In some embodiments, the invention relates to thetreatment or prevention of PML using protein channel antagonists. Insome embodiments, the invention relates to the treatment or preventionof PML using compounds that are structurally related to endosulfan.

In some embodiments, the invention relates to the use of candesartancilextil, and compounds with the same mode of action as candesartancilextil, to prevent or treat DNA viral infection or proliferation(e.g., to reduce the risk of DNA viral infection or proliferation).Candesartan cilextil can act as an angiotensin II antagonist. In someembodiments, the invention relates to the treatment or prevention of PMLusing angiotensin II antagonists. In some embodiments, the inventionrelates to the treatment or prevention of PML using compounds that arestructurally related to candesartan cilextil.

In some embodiments, the invention relates to the use of fusidic acid,and compounds with the same mode of action as fusidic acid, to preventor treat DNA viral infection or proliferation (e.g., to reduce the riskof DNA viral infection or proliferation). In some embodiments, theinvention relates to the treatment or prevention of PML usingantibiotics. In some embodiments, the invention relates to the treatmentor prevention of PML using compound that interfere with bacterialprotein synthesis. In some embodiments, the invention relates to thetreatment or prevention of PML using compounds that are structurallyrelated to fusidic acid. In some embodiments, compounds that arestructurally related to fusidic acid are steroid derivatives. In someembodiments, compounds that are structurally related to fusidic acid are4-estren-3-beta 17 beta-diol 17-acetate, 5-beta-pregnan-3-alpha 6-alpha20-beta-triol 20-acetate and 4-pregen-3-beta 20-beta-diol 20-acetate.

In some embodiments, the invention relates to the treatment orprevention of PML using compounds that are structurally related tocandesartan cilextil.

In some embodiments, the invention relates to the use of mefenamic acid,and compounds with the same mode of action as mefenamic acid, to preventor treat DNA viral infection or proliferation (e.g., to reduce the riskof DNA viral infection or proliferation). Mefenamic acid is a drug thatbelong to a class of NSAIDs. In some embodiments, the invention relatesto the treatment or prevention of PML using NSAIDs.

In one aspect, the invention provides methods of treatment of a subjectinfected with a DNA virus. In some embodiments, the DNA virus is a JCvirus. In some embodiments, the DNA virus is a BK virus. In someembodiments, the invention provides methods of treatment or preventionfor subjects having, or at risk of developing, progressive multifocalleukoencephalopathy (PML).

It should be appreciated that the inhibitory compounds or compositionsdescribed herein may be used to reduce or suppress DNA replication ofDNA viruses (e.g., JC virus, BK virus, or any other DNA virus).

The human genome is exposed to and may acquire many viruses during thelifetime of an individual. One group of viruses the genome is exposed toare DNA viruses. DNA viruses include papova viruses and herpes viruses.Examples of papova viruses include, but are not limited to SV40, humanor bovine papilloma virus (HPV or BPV), polyoma virus and humanSV40-like viruses such as BK (BKV) or JC (JCV). Examples of herpesviruses include, but are not limited to herpes simplex virus (HSV),cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella or chickenpoxvirus, herpes zoster or shingles virus. Exposure to a DNA virus is oftenasymptomatic and a subject may not be aware that it has been exposed toa DNA virus. Exposure to DNA viruses often leads to integration of theDNA virus in the human genome. In a subject with a healthy immunesystem, the immune system will generally suppress the proliferation ofthe virus. However, in immuno-compromised subjects or in subjectsundergoing immune treatment, DNA viruses may be expressed andproliferate resulting in the development of disease.

In one aspect, the current invention provides methods of treatingsubjects infected with the JC polyomavirus (JCV). Primary infection withJCV can occur asymptomatically during childhood (Padgett et al., 1971Lancet., 1257-1260). JCV is then disseminated throughout the body,probably through viraemia (Ikegaya et al., 2004, Archives of Virology149, 1215-1220). It is thought that JCV persists mostly in brain andrenal tissue. While infection by JCV is asymptomatic in most subjects,infection can result in serious conditions (such as PML) and even death.PML is an extremely debilitating demyelination disease of the centralnervous system. PML is generally characterized by neurological deficitsthat progress rapidly, typically without signs of intracranial pressure,including hemiparesis, cognitive disturbance, visual field deficits,ataxia, aphasia, cranial nerve deficits and sensory deficits. Patientswho have PML typically deteriorate rapidly and death commonly occurswithin 6 months of diagnosis (Demeter L M. J C, B K, and otherpolyomaviruses; progressive multifocal leukoencephalopathy. In Mandell GL, Bennett J E, Dolin, eds. Mandell, Douglas and Bennett's Principlesand Practice of Infectious Diseases, 4th edition, Vol. 2. New York,N.Y.: Churchill Livingstone; 1995: 1400-1406). Subjects most susceptibleto PML are subjects that are immuno-compromised (e.g., AIDS patients) orsubjects undergoing treatment with immunosuppressants (for instanceafter organ transplant or to treat an inflammation related conditionsuch as multiple sclerosis or rheumatoid arthritis).

PML has been reported to be associated with certain JCV variants thathave acquired sequence variations relative to a “wild type” JCV sequence(Zheng et al., 2004, Microbes Infect., 6, 596-603). A “wild type” JCVsequence is used herein to refer to the sequence of any of thearchetypes of JCV found in healthy subjects not having PML, and/or notbeing at risk for PML. In some embodiments, a consensus “wild type”reference sequence may be an average of sequences found in a group ofhealthy subjects. Interestingly, in subjects having PML, a JCV with anumber of sequence variations was isolated from the brain, while a wildtype variant was isolated from the urine of the same individual (Yogo etal., 2004, Rev Med Virol 14, 179-191). Accordingly, a subject may beinfected with several different versions of JCV concurrently. In someembodiments of the present invention, a subject infected with a JCVvariant associated with PML is a subject at risk for developing PML.

In one aspect, the current invention provides methods of treatingsubjects infected with the BK virus. Infection with BK virus is thoughtto be widespread but mostly asymptomatic. The lung, eye, liver, brainand kidney are sites of BK virus-associated disease. Infection in thekidney can lead to hemorrhage, non-hemorrhagic cystitis, uretericstenosis and nephritis. Infection in the CNS has been associated withencephalitis and Guillian-Barre syndrome. An overview of diseasesassociated with BK virus can be found for instance in Reploeg et al.(Clinical Infectious Diseases 2001; 33: 191-202), the contents of whichare incorporated herein by reference. In some embodiments, the currentinvention provides methods of treating subjects at risk for infection byBK virus. Subjects that are immuno-compromised or receivingimmunosuppressive agents are at an increased risk for BK virus infectionresulting in pathogenesis. Pathogenic BK virus infection can beespecially problematic in patients undergoing a kidney transplant, whichis often accompanied with the use of immunosuppressive agents.

It should be appreciated that an immunosuppressive agent may increasethe susceptibility of a subject to the progression or flare up of alatent microbial infection or to the contraction of a new microbialinfection. In some embodiments, the microbial infection is infection bya DNA virus. In some embodiments, the microbial infection is infectionby JCV, which causes PML. In some embodiments, the microbial infectionis infection by BK virus. In some embodiments, subjects that areimmuno-compromised (e.g., AIDS patients) or subjects undergoingtreatment with immunosuppressants are subjects art risk for PML.Subjects at risk for PML include subjects that may receive or havereceived treatment with one or more immunosuppressive agents (alsocalled immunosuppressants). In some cases, the immunosuppressive agentis administered to the subject for treatment of a disease or condition,including one or more of the following non-limiting examples: cancer,organ transplant, tissue transplant, an inflammatory condition ordisease, multiple sclerosis (MS), arthritis, or any combination thereof.In some embodiments, the immunosuppressive agent is an anti-VLA-4antibody (e.g., natalizumab). In some cases, an at-risk subject testspositive for the presence of a JCV nucleic acid or a JCV polypeptide. Inother cases, the at-risk subject does not test positive for the presenceof a JCV nucleic acid or a JCV polypeptide.

Currently, there is no specific antiviral therapy that has been proveneffective for treatment of JCV infection, and current treatment ofimmuno-compromised subjects infected with JCV, or at risk for JCVinfection, is primarily focused on improving the function of the immunesystem in general. Similarly, in subjects infected with JCV or at riskfor JCV infection that are receiving immuno-therapy, current treatmentmethods of the JCV infection are limited to termination or decrease indose of the immuno-therapy. Therapies currently used to treat PML relyon enhancement of the immune response, e.g., HAART (Highly ActiveAnti-Retroviral Therapy) in HIV positive patients (e.g., Marzocchetti etal., 2005, J Clin Microbiology 434: 4175-4177) or decrease in the amountof immunosuppressive drugs in subjects receiving those drugs (e.g.,transplant patients). Methods of treatment of JCV include, but are notlimited to, IFN-alpha, cytarabine, and cidofovir. 5HT2a blockers mayalso be used as a treatment and are currently under study.

In some embodiments, a subject receiving an immunosuppressive agent maybe treated with compositions comprising one or more of the compounds ofTable 1. In some embodiments, the treatment is prophylactic, e.g., thesubject receiving immunosuppressants may be treated with compositionscomprising one or more compounds of Table 1, even if the subject has notbeen shown to be infected with a DNA virus, including JCV or BKV, anddoes not show any symptoms associated with PML.

In some embodiments, a subject may be treated by combiningimmunosuppressive treatment with treatment against DNA virus infectionor the development of diseases associated with DNA virus infection,including PML. In one aspect, the invention comprises methods oftreatment comprising the administration of one or more compounds ofTable 1 and an immunosuppressant. In one aspect, the invention comprisesmethods of treatment comprising the administration of one or morecompounds of Table 1 and natalizumab. In one aspect, the inventioncomprises methods of treatment comprising the administration of acomposition comprising one or more compounds of Table 1 and ananti-VLA-4 antibody (e.g., natalizumab).

In some embodiments, administration of one or more compounds of Table 1may be combined with methods to remove or partially remove animmunosuppressant from the bloodstream of a subject. In someembodiments, the immunosuppressant may be removed from the bloodstreamprior to administration of one or more compounds of Table 1. In someembodiments, the one or more compounds of Table 1 may be combined with acompound that can bind to an immunosuppressant resulting in an increasedclearance of the immunosuppressant from the bloodstream. In someembodiments, the method of removal or partial removal of theimmunosuppressant from the bloodstream of a subject is plasma exchange(PLEX). In plasma exchange blood is taken from the body and plasmacontaining the immunosuppressant is removed from the blood by a cellseparator. Blood can be removed from the body in batches or it can beremoved in a continuous flow mode, with the latter allowing for thereintroduction of the processed blood into the body. The removed plasmacomprising the immunosuppressants will be discarded and the patient willreceive donor plasma or saline with added proteins in return. In someembodiments, multiple rounds of plasma exchange may be needed to removethe immunosuppressant from the blood or to lower the level of theimmunosuppressant in the blood to an acceptable level. Methods of plasmaexchange are well known in the art and are described for instance inU.S. Pat. No. 6,960,178.

The term “immunomodulatory” treatment or therapy refers to theadministration of one or more compounds that modulate (e.g., upregulateor downregulate) one or more aspects of a subject's immune system. Insome embodiments, an immunomodulatory treatment or therapy involves theadministration of one or more immunosuppressive agents to a subject. Insome embodiments, an immunomodulatory treatment or therapy downregulatesthe immune system of a subject. Accordingly, in some embodiments, animmunomodulatory therapy is an immunosuppressive therapy. In someembodiments, an immunomodulatory treatment or therapy downregulates theimmune system of a subject. It should be appreciated that animmunomodulatory treatment or therapy (e.g., an immunosuppressivetreatment or therapy) as used herein also may be referred to as animmunotherapy in the context of methods described herein.

The term “immunosuppressive agent” as used herein refers to substancesthat act to suppress or mask the immune system of a subject beingtreated herein. Immunosuppressive agents may be substances that suppresscytokine production, down-regulate or suppress self-antigen expression,or mask the MHC antigens. Examples of such agents include2-amino-6-aryl-5-substituted pyrimidines (see e.g., U.S. Pat. No.4,665,077); nonsteroidal anti-inflammatory drugs (NSAIDs); ganciclovir,tacrolimus, glucocorticoids such as cortisol or aldosterone,anti-inflammatory agents such as a cyclooxygenase inhibitor, a5-lipoxygenase inhibitor, or a leukotriene receptor antagonist; purineantagonists such as azathioprine or mycophenolate mofetil (MMF);alkylating agents such as cyclophosphamide; bromocryptine; danazol;dapsone; glutaraldehyde (which masks the MHC antigens, as described inU.S. Pat. No. 4,120,649); anti-idiotypic antibodies for MHC antigens andMHC fragments; cyclosporin A; steroids such as corticosteroids orglucocorticosteroids or glucocorticoid analogs, e.g., prednisone,methylprednisolone, and dexamethasone; dihydrofolate reductaseinhibitors such as methotrexate (oral or subcutaneous);hydroxycloroquine; sulfasalazine; leflunomide; cytokine or cytokinereceptor antagonists including anti-interferon-alpha, -beta, or -gammaantibodies, anti-tumor necrosis factor-alpha antibodies (infliximab oradalimumab), anti-TNF-alpha immunoahesin (etanercept), anti-tumornecrosis factor-beta antibodies, anti-interleukin-2 antibodies andanti-IL-2 receptor antibodies; anti-LFA-1 antibodies, includinganti-CD11a and anti-CD18 antibodies; anti-CD20 antibodies (e.g.,rituximab, for example available under the trademark RITUXAN); anti-L3T4antibodies; anti-VLA-4 antibodies (e.g., natalizumab); heterologousanti-lymphocyte globulin; pan-T antibodies, for example anti-CD3 oranti-CD4/CD4a antibodies; soluble peptide containing a LFA-3 bindingdomain (WO 90/08187 published Jul. 26, 1990); streptokinase; TGF-beta;streptodornase; RNA or DNA from the host; FK506; RS-61443;deoxyspergualin; rapamycin; T-cell receptor (Cohen et al., U.S. Pat. No.5,114,721); T-cell receptor fragments (Offner et al., Science, 251:430432 (1991); WO 90/11294; Ianeway, Nature, 341: 482 (1989); and WO91/01133); and T cell receptor antibodies (EP 340,109) such as T10B9.However, subjects receiving other immunosuppressive agents are alsoencompassed by the invention.

Accordingly, immunosuppressive agents may be drugs that inhibit orprevent certain aspects of the immune system. Immunosuppressive agentsmay be drugs that are used in immunomodulatory (e.g., immunosuppressive)therapy, for example, to prevent the rejection of transplanted organs ortissues (e.g., bone marrow, heart, kidney, liver), to treat autoimmunediseases or diseases that are suspected of being associated with anautoimmune reaction (e.g., rheumatoid arthritis, multiple sclerosis,myasthenia gravis, systemic lupus erythematosus, an inflammatory boweldisease or syndrome, including, for example, Crohn's disease, ulcerativecolitis, and pemphigus), or to treat a non-autoimmune inflammatorydisease or condition (e.g., for term allergic asthma control).

Immunosuppressive agents may be classified into five general categories:glucocorticoids, cytostatics, antibodies, drugs acting on immunophilins,and other drugs. Glucocorticoids may include drugs that suppresscell-mediated immunity (e.g., by inhibiting IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-8, and/or TNF-γ. However, glucocorticoids also maysuppress humoral immunity. Cytostatics may include drugs that inhibitcell division (e.g., agents that inhibit T cell and/or B cellproliferation). In some embodiments, cytostatics may be alkylatingagents such as nitrogen mustards (e.g., cyclophosphamide), nitrosureas,platinum compounds, and others. In certain embodiments,immunosuppressive agents may be antimetabolites such as folic acidanalogs (e.g., methotrexate), purine analogs (e.g., azathiprine,mercaptopurine), pyrimidine analogs, or protein synthesis inhibitors. Animmunosuppressive agent also may be a cytotoxic antibiotic such asdactinomycin, an anthracycline, mytomicin C, bleomycin, or mithramycin.Cytostatic antibodies may be polyclonal or monoclonal antibodies thatinhibit one or more aspects of the immune system (e.g., that inhibit Tlymphocytes). Non-limiting examples of immunosuppressive polyclonalpreparations include Atgam (R), obtained from horse serum, andThymoglobuline (R), obtained from rabbit serum. In some embodiments,monoclonal antibodies may be IL-2 receptor (CD25) and/or CD3 directedantibodies. Non-limiting examples of immunosuppressive monoclonalantibodies include T-cell receptor directed antibodies (e.g., murorab,an anti-CD3 antibody), and/or IL-2 receptor directed antibodies (e.g.,basiliximab (Simulect (R)) and daclizumab (Zenapax (R)). Cytostaticdrugs acting on immunophilins may include cyclosporin, tacrolimus (e.g.,Prograf), and/or sirolimus (e.g., Rapamune, or Rapamycin). Othercytostatic drugs may include interferons, opioids, TNF binding proteins(e.g., infliximab (e.g., Remicade), etanercept (e.g., Embrel), oradalimumab (e.g., Humira)), myophenolate, and/or small biological agents(e.g., FTY720, or myriocin).

Immunosuppressive agents that may be used to reduce the risk oftransplant rejection include, but are not limited to: calcineurininhibitors (e.g., Cyclosporin or Tacrolimus), mTOR inhibitors (e.g.,Sirolimus or Everolimus), anti-proliferatives (e.g., Azathioprine,Mycophenolic acid), corticosteroids (e.g., prednisolone orhydrocortisone), and/or antibodies (e.g., monoclonal anti-IL-2Ralphareceptor antibodies such a basiliximab or daclizumab or polyclonalanti-T-cell antibodies such as anti-thymocyte globulin (ATG) oranti-lymphocyte globulin (ALG)).

In some embodiments, the immunosuppressant is a VLA-4 binding antibodylike natalizumab (also known as TYSABRI®). In some embodiments, a VLA-4binding antibody is an IgG antibody (e.g., an IgG4 antibody). In someembodiments, a VLA-4 binding antibody is a polyclonal or monoclonalantibody. In some embodiments, a VLA-4 binding antibody is a humanizedversion of a murine antibody. Natalizumab and related VLA-4 bindingantibodies are described, e.g., in U.S. Pat. No. 5,840,299. mAb 21.6 andHP1/2 are exemplary murine monoclonal antibodies that bind VLA-4.Natalizumab is a humanized version of murine mAb 21.6 (see, e.g., U.S.Pat. No. 5,840,299). A humanized version of HP1/2 has also beendescribed (see, e.g., U.S. Pat. No. 6,602,503). Several additional VLA-4binding monoclonal antibodies, such as HP2/1, HP2/4, L25 and P4C2, aredescribed (e.g., in U.S. Pat. No. 6,602,503; Sanchez-Madrid et al., 1986Eur. J. Immunol., 16:1343-1349; Hemler et al., 1987 J. Biol. Chem.2:11478-11485; Issekutz and Wykretowicz, 1991, J. Immunol., 147: 109(TA-2 mab); Pulido et al., 1991 J. Biol. Chem., 266(16):10241-10245; andU.S. Pat. No. 5,888,507). Many useful VLA-4 binding antibodies interactwith VLA-4 on cells, e.g., lymphocytes, but do not cause cellaggregation. However, other anti-VLA-4 binding antibodies have beenobserved to cause such aggregation. HP1/2 does not cause cellaggregation. The HP1/2 MAb (Sanchez-Madrid et al., 1986 Eur. J.Immunol., 16:1343-1349) has an extremely high potency, blocks VLA-4interaction with both VCAM1 and fibronectin, and has the specificity forepitope B on VLA-4. This antibody and other B epitope-specificantibodies (such as B1 or B2 epitope binding antibodies; Pulido et al.,1991 J. Biol. Chem., 266(16):10241-10245) represent one class of usefulVLA-4 binding antibodies.

An exemplary VLA-4 binding antibody has one or more CDRs, e.g., allthree HC CDRs and/or all three LC CDRs of a particular antibodydisclosed herein, or CDRs that are, in sum, at least 80, 85, 90, 92, 94,95, 96, 97, 98, or 99% identical to natalizumab. In one embodiment, theH1 and H2 hypervariable loops have the same canonical structure as thoseof natalizumab. In some embodiments, the L1 and L2 hypervariable loopshave the same canonical structure as natalizumab. In some embodiments,the amino acid sequence of the HC and/or LC variable domain sequence isat least 70, 80, 85, 90, 92, 95, 97, 98, 99, or 100% identical to theamino acid sequence of the HC and/or LC variable domain of natalizumab.The amino acid sequence of the HC and/or LC variable domain sequence candiffer by at least one amino acid, but no more than ten, eight, six,five, four, three, or two amino acids from the corresponding sequenceof, natalizumab. For example, the differences may be primarily orentirely in the framework regions. The amino acid sequences of the HCand LC variable domain sequences can be encoded by a sequence thathybridizes under high stringency conditions to a nucleic acid sequencedescribed herein or one that encodes a variable domain or to a nucleicacid encoding an amino acid sequence described herein. In oneembodiment, the amino acid sequences of one or more framework regions(e.g., FR1, FR2, FR3, and/or FR4) of the HC and/or LC variable domainare at least 70, 80, 85, 90, 92, 95, 97, 98, 99, or 100% identical tocorresponding framework regions of the HC and LC variable domains ofnatalizumab. In some embodiments, one or more heavy or light chainframework regions (e.g., HC FR1, FR2, and FR3) are at least 70, 80, 85,90, 95, 96, 97, 98, or 100% identical to the sequence of correspondingframework regions from a human germline antibody.

Calculations of “homology” or “sequence identity” between two sequences(the terms are used interchangeably herein) are performed as follows.The sequences are aligned for optimal comparison purposes (e.g., gapscan be introduced in one or both of a first and a second amino acid ornucleic acid sequence for optimal alignment and non-homologous sequencescan be disregarded for comparison purposes). The optimal alignment isdetermined as the best score using the GAP program in the GCG softwarepackage with a Blossum 62 scoring matrix with a gap penalty of 12, a gapextend penalty of 4, and a frameshift gap penalty of 5. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences. The skilled artisan willrealize that conservative amino acid substitutions may be made in VLA-4binding antibodies to provide functionally equivalent variants of theseantibodies. As used herein, a “conservative amino acid substitution”refers to an amino acid substitution that does not alter the relativecharge or size characteristics of the protein in which the amino acidsubstitution is made. Variants can be prepared according to methods foraltering polypeptide sequence known to one of ordinary skill in the artsuch as are found in references that compile such methods, e.g.,Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N. Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel,et al., eds., John Wiley & Sons, Inc., New York. Exemplary functionallyequivalent variants of VLA-4 binding antibodies include conservativeamino acid substitutions of in the amino acid sequences of proteinsdisclosed herein. Conservative substitutions of amino acids includesubstitutions made amongst amino acids within the following groups: (a)M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and(g) E, D.

In some embodiments, the methods of treatment or prevention compriseadministering a composition comprising one or more compounds of Table 1and one or more known or putative anti-viral compounds or compoundsdisplaying anti-viral activity. Known or putative anti-viral compoundsare compounds that suppress or inhibit viral infection, viralproliferation and/or the development of disease associated with viralinfection. Anti-viral drugs can be classified as targeting one of thelife cycle stages of the virus. One category of anti-viral drugs arebased on interfering with viral entry. A virus binds to a specificreceptor to infiltrate a target cell. Viral entry can be suppressed byblocking of the viral entry way. Anti-viral drugs that have this mode ofaction are anti-receptor antibodies, natural ligands of the receptor andsmall molecules that can bind to the receptor. A second category ofantiviral drugs are compounds that suppress viral synthesis. Antiviraldrugs that have this mode of action are nucleoside analogues that aresimilar to the DNA and RNA building blocks but deactivate the proteinmachinery (e.g., reverse transcriptase or DNA polymerase) used toreplicate the virus. Other drugs are targeted at blocking thetranscription factors of viral DNA, ribozymes, which can interfere withthe production of viral DNA. Other drugs target viral RNA fordestruction, including siRNAs and antisense nucleic acids against viralnucleic acid sequences. Yet another class of antiviral drugs are drugsthat can interfere with the function of virus specific proteins. Thisclass includes the HIV protease inhibitors. Antiviral drugs also includedrugs directed at the release stage if the virus. This category of drugsinclude compounds that interfere with the proteins necessary to buildthe viral particles. Another class of antiviral drugs are drugs thatstimulate the immune system in targeting viral infection. Drugs thatfall in this class are interferons, which inhibit viral synthesis ininfected cells. and antibodies that can target an infected cell fordestruction by the immune system. Other anti-viral agents are describedin U.S. Pat. Nos. 6,130,326, and 6,440,985, and published US patentapplication 20020095033. Accordingly, it should be appreciated thatcompounds identified herein have antiviral activity and may act throughany antiviral mechanism described above. In some embodiments, compoundsidentified herein inhibit or suppress viral replication (e.g., viral DNAreplication).

The anti-viral activity of a compound may be assayed in an in vitro cellbased assay. Anti-viral activity may result from i) the interaction of acompound with the virus to prevent infection of a cell or to preventreplication, development, and/or proliferation of the virus afterinfection, ii) the effect of a compound on a cell to prevent infectionby the virus or to prevent replication, development, and/orproliferation of the virus after infection, or iii) any other mechanism,or any combination thereof. Regardless of the mode of action, acomposition of the invention may have anti-viral activity if it reducesthe percentage or number of infected cells in a cell-based assay. Insome embodiments, a compound (or a combination of two or more compounds)has anti-viral activity when it reduces the percentage or number ofinfected cells by at least 20%, at least 30%, at least 40%, at least50%, or more (e.g., in a cell-based assay). In some embodiments, acompound has anti-viral activity when it reduces the amount of viralnucleic acids within a cell. In certain embodiments, a compound inhibitsthe replication of viral nucleic acids within a cell (e.g., a compoundreduces the amount of viral replication by about 5%, about 10%, about20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,about 90%, or higher or lower or intermediate percentages of reduction).It should be appreciated that a reduction in viral replication may bemeasured using a cellular assay and measuring the amount of viral DNA orthe rate of viral DNA replication over time (or any other measure ofviral replication) in the presence of a compound and comparing it to theviral replication in the absence of the compound or in the presence of acontrol compound.

It should be appreciated that certain compositions may effectivelyinhibit viral activity in certain cell types and not others. Acomposition of the invention may be useful if it is effective in certaincell types regardless of whether it is active in all cell types (or evenin more than one cell type). For example, compositions of the inventionmay be compositions that are effective at least in one or more neuralcells, for example, one or more cells of the central nervous system(e.g., glial cells, astrocytes, etc.). It should be appreciated that Insome embodiments, non-cytotoxic compounds (or compounds that selectivelykill infected cells) are used in compositions and methods of theinvention.

In some embodiments, the methods of treatment or prevention compriseadministering a composition comprising one or more compounds of Table 1and administering a vaccine against a DNA virus. A vaccine is defined asa pharmaceutical composition that when administered to a subject in aneffective amount stimulates the production of protective antibody orprotective T-cell response. In some embodiments, the vaccine is proteinvaccine comprising one or more polypeptide sequences encoded by a DNAvirus sequence. In some embodiments, the vaccine is a nucleic acidvaccine comprising DNA viral nucleic acids. Administration regimes forvaccines are known to a person of ordinary skill in the art. In someembodiments, ranges of amounts of polypeptide vaccines for prophylaxisof DNA viral infection are from 0.01 to 100 microgram/dose, for example0.1 to 50 microgram/dose. Several doses may be needed per subject inorder to achieve a sufficient immune response and subsequent protectionagainst DNA viral infection (e.g., “immunizing” a subject). The term“immunizing” refers to the ability of a substance to cause a humoraland/or cellular response in a subject, whether alone or when linked to acarrier, in the presence or absence of an adjuvant, and also refers toan immune response that blocks the infectivity, either partially orfully, of an infectious agent.

In some embodiments, the methods of treatment or prevention compriseadministering one or more compounds of Table 1 and administering anantibody against a DNA virus. In some embodiment the methods oftreatment or prevention comprise administering one or more compounds ofTable 1 and administering an antibody against JCV. Antibodies may beused in therapy to treat subjects with PML and/or to prevent infectionby and/or suppress the activity of JCV and other DNA viruses. Suitableantibodies or fragments thereof may be selected for the ability to bindone or more polypeptides encoded by a DNA virus, including JCV. Theantibody or antigen-binding fragment thereof may be an IgG1, IgG2, IgG3,IgG4, IgM, IgA1, IgA2, IgAsec, IgD, IgE or may have an immunoglobulinconstant and/or variable domain of an IgG1, IgG2, IgG3, IgG4, IgM, IgA1,IgA2, IgAsec, IgD or IgE. In some embodiments, the antibody is abispecific or multispecific antibody. In some embodiments, the antibodyis a recombinant antibody, a polyclonal antibody, a monoclonal antibody,a humanized antibody or a chimeric antibody, or a mixture of these. Insome embodiments, the antibody is a human antibody, e.g., a humanmonoclonal antibody, polyclonal antibody or a mixture of monoclonal andpolyclonal antibodies. Antigen-binding fragments may include a Fabfragment, a F(ab′)₂ fragment, and/or a F_(v) fragment CDR3. Antibodiescan be raised against a full length DNA virus protein or JCV protein oragainst polypeptides variants comprising a partial sequence of a DNAvirus protein or JCV protein. Antibodies can be generated by injectingan animal, for example a rabbit or goat or mouse, with the antigen(e.g., a polypeptide of a DNA virus protein or JCV protein). In order toprepare polyclonal antibodies, fusion proteins containing a polypeptideof a DNA virus protein or JCV protein can be synthesized in bacteria byexpression of corresponding DNA sequences in a suitable cloning vehicle.The fusion protein can then be purified, coupled to a carrier proteinand mixed with Freund's adjuvant (to help stimulate the antigenicresponse by the rabbits) and injected into rabbits or other laboratoryanimals. Alternatively, the polypeptides can be isolated from culturedcells expressing the protein. Following booster injections at bi-weeklyintervals, the rabbits or other laboratory animals are then bled and thesera isolated. The sera can be used directly or purified prior to use,e.g., by methods such as affinity chromatography, Protein A-Sepharose,Antigen Sepharose, Anti-mouse-Ig-Sepharose. The sera can then be used toprobe protein extracts run on a polyacrylamide gel to identify the DNAvirus or JCV polypeptides. Alternatively, synthetic DNA virus or JCVpolypeptides can be made and used to inoculate animals. To producemonoclonal DNA virus or JCV antibodies, mice are injected multiple times(see above), the mice spleens are removed and resuspended in a phosphatebuffered saline (PBS). The spleen cells serve as a source oflymphocytes, some of which produce antibodies of the appropriatespecificity. These are then fused with a permanently growing myelomapartner cell, and the products of the fusion are plated into a number oftissue culture wells in the presence of a selective agent such as HAT.The wells are then screened by ELISA to identify those containing cellsexpressing useful antibody. These are then freshly plated. After aperiod of growth, these wells are again screened to identifyantibody-producing cells. Several cloning procedures are carried outuntil over 90% of the wells contain single clones which are positive forantibody production. From this procedure a stable line of clones isestablished to produce the antibody. A monoclonal antibody can then bepurified by affinity chromatography using Protein A Sepharose,ion-exchange chromatography, as well as variations and combinations ofthese techniques (See e.g., U.S. Pat. No. 6,998,467). For antibodies tobe used in therapy in humans, they may be ‘humanized’. Humanization ofantibodies involves replacing native mouse sequences with humansequences to lower the chance of an immune response once the therapeuticantibody is introduced into humans. In some embodiments, humanantibodies (e.g., identified from libraries of human antibodies) may beused.

In some embodiments, the compounds that prevent or treat DNA viralinfection or proliferation have an IC₅₀<100 μM, an IC₅₀<20 μM, anIC₅₀<10 μM, an IC₅₀<5 μM, or an even lower IC₅₀. The IC₅₀ (InhibitoryConcentration) is defined herein as the inhibitory concentration atwhich 50% of JC viral infection is inhibited. In some embodiments, thecompounds that prevent or treat DNA viral infection or proliferationhave a TC₅₀>5 μM, a TC₅₀>20 μM, a TC₅₀>50 μM, a TC₅₀>100 μM, or an evenhigher TC₅₀. The TC₅₀ (cytoToxic Concentration) is defined herein as theconcentration of the inhibitor at which 50% of cells are killed. In someembodiments, the compounds that prevent or treat DNA viral infection orproliferation have an IC₅₀/TC₅₀<5, an IC₅₀/TC₅₀<1, an IC₅₀/TC₅₀<0.5, anIC₅₀/TC₅₀<0.2, an IC₅₀/TC₅₀<0.2 or an even lower IC₅₀/TC₅₀. A compoundwith a lower IC₅₀/TC₅₀ potentially has a larger potential therapeuticwindow, as a lower IC₅₀/TC₅₀ correlates to a lower IC₅₀ (inhibitoryconcentration) and a higher TC₅₀ (cytotoxic concentration).

In some embodiments, the compounds that prevent or treat DNA viralinfection or proliferation, upon administration have a concentration inthe target tissue of at least 0.5×IC₅₀, at least 1×IC₅₀, at least2×IC₅₀, at least 3×IC₅₀, at least 10×IC₅₀ or higher. In someembodiments, the compounds that prevent or treat DNA viral infection orproliferation, upon administration have a concentration in the targettissue of less than 1×TC₅₀, less than 0.5×TC₅₀, less than 0.2×TC₅₀, lessthan 0.1×TC₅₀, less than 0.01×TC₅₀ or lower. In some embodiments, thecompounds that prevent or treat DNA viral infection or proliferation,upon administration have an IC₅₀/TC₅₀ ratio in the target tissue of lessthan <5, an IC₅₀/TC₅₀<1, an IC₅₀/TC₅₀<0.5, an IC₅₀/TC₅₀<0.2, anIC₅₀/TC₅₀<0.2 or an even lower IC₅₀/TC₅₀.

Target tissue, as used herein, embraces any tissue in a subject,including but not limited to kidney, brain, liver, spleen, bone marrow,intestine, stomach. In some embodiments, the target tissue is braintissue or CNS. In some embodiments, the target tissue is the kidney. Insome embodiments, the compounds that prevent or treat DNA viralinfection or proliferation have a high plasma level upon administration.In some embodiments, the compounds that prevent or treat DNA viralinfection or proliferation, wherein the DNA viral infection is locatedin the kidney or is expected to target the kidney, have a high plasmalevel upon administration.

In some embodiments, infection with a DNA virus is characterized by, orhas a potential to result in, infection of the brain, including the CNSand CSF. In some embodiments, one or more of the compounds of Table 1are administered to a subject that has a DNA virus infection of thebrain, is at risk for infection of the brain by a DNA virus, or has adisease associated with a DNA virus infection of the brain (e.g., PML).In some embodiments, one or more of the compounds of Table 1 areadministered in conjunction with an agent that targets or facilitatesdelivery of the compounds across the blood brain barrier.

As used herein a “blood-brain barrier targeting agent” is a molecule orcompound that is capable of crossing the blood-brain barrier and can beused to deliver a therapeutic composition of the invention across theblood-brain barrier and into the CNS. As used herein a “CNS-celltargeting agent” is a molecule or compound that delivers a compositionof the invention to a region of the CNS or to, or into, a CNS cell typeonce the composition is inside the blood-brain barrier. It will beunderstood that in some embodiments of the invention, a therapeuticcomposition may be attached to one or more blood-brain targeting agentsand may be also attached to one or more CNS cell targeting agents. Thusthere may be more than one type of blood-brain barrier targeting agentand/or more than one CNS cell targeting agent attached to a carrier ofthe invention. In some embodiments, one or more of the compounds ofTable 1 may be packaged in a carrier (e.g., a liposome) to facilitate ortarget delivery to the brain and/or CNS.

In some embodiments, one or more of the compounds of Table 1 may beadministered directly to a tissue infected by, or suspected of beinginfected by, the DNA virus (e.g., JCV, BCV, or other DNA virus). In someembodiments, one or more of the compounds of Table 1 may be administereddirectly to the brain. In some embodiments, one or more of the compoundsof Table 1 may be administered through intrathecal injection. Anintrathecal injection is an injection into the spinal canal whichfacilitates direct delivery of a compound to the CNS and the brain,thereby circumventing the blood brain barrier. In some embodiments,intrathecal injection is performed as a method of administration ofcompounds that have a low oral administration.

The phrase “therapeutically-effective amount” as used herein means thatamount of a compound, material, or composition comprising one or morecompounds of the present invention which are effective for producingsome desired therapeutic effect in a subject at a reasonablebenefit/risk ratio applicable to any medical treatment. Accordingly, insome embodiments, a therapeutically effective amount prevents,minimizes, or reverses disease progression associated with infectionwith a DNA virus (including JCV, BCV, or other DNA virus). Diseaseprogression can be monitored by clinical observations, laboratory andimaging investigations apparent to a person skilled in the art. Atherapeutically effective amount can be an amount that is effective in asingle dose or an amount that is effective as part of a multi-dosetherapy, for example an amount that is administered in two or more dosesor an amount that is administered chronically.

The effective amount of any one or more compounds may be from about 10ng/kg of body weight to about 1000 mg/kg of body weight, and thefrequency of administration may range from once a day to once a month.However, other dosage amounts and frequencies also may be used as theinvention is not limited in this respect. A subject may be administeredone or more compounds described herein in an amount effective to treator prevent infection with a DNA virus. As used herein, a treatment maybe prophylactic and/or therapeutic. In some embodiments, a treatment mayinclude preventing viral infection and/or proliferation. In certainembodiments, a treatment may include inhibiting and or reducing viralinfection and/or proliferation. It should be appreciated that the termspreventing and/or inhibiting may be used to refer to a partialprevention and/or inhibition (e.g., a percentage reduction, for exampleabout 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about60%, about 70%, about 80%, about 90%, or higher or lower or intermediatepercentages of reduction). However, in some embodiments, a prevention orinhibition may be complete (e.g., a 100% reduction or about a 100%reduction based on an assay).

In some embodiments, the effective amount is tissue specific. In someembodiments, the effective amount is an amount of one or more compoundsof Table 1 that results in prevention or inhibition of viral activity ina specific tissue (e.g., viral proliferation in that tissue). In someembodiments, the effective amount is an amount of one or more compoundsof Table 1 that results in prevention or inhibition of viral activity inthe brain. In some embodiments, the effective amount is an amount of oneor more compounds of Table 1 that results in about 5%, about 10%, about20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,about 90%, or higher or lower or intermediate percentages of inhibitionof viral activity in the brain. In some embodiments, the effectiveamount is an amount of one or more compounds of Table 1 that results inabout 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about60%, about 70%, about 80%, about 90%, or higher or lower or intermediatepercentages of reduction of viral DNA replication in a cell (e.g., asmeasured in a cellular assay) when compared to the percentage reductionof viral DNA replication in the absence of a compound (or when the cellis exposed to a control compound that does not have antiviral activity,e.g., saline or vehicle control). In some embodiments, the biologicalsample concentration of one or more compounds of Table 1 is 1 nM, 10 nM,100 nM, 1 μM, 10 μM, 100 μM, 1 mM, 10 mM, 100 mM after administration ofthe one or more compounds of Table 1. In some embodiments, thebiological sample concentration of one or more compounds of Table 1 isat least 10 μM after administration of the one or more compounds ofTable 1. In some embodiments, the concentration of one or more compoundsof Table 1 in a specific tissue is 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100μM, 1 mM, 10 mM, 100 mM after administration of the one or morecompounds of Table 1. In some embodiments, the concentration of one ormore compounds of Table 1 in a specific tissue is at least 10 μM afteradministration of the one or more compounds of Table 1. In someembodiments, the concentration of one or more compounds of Table 1 inthe brain is 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, 10 mM, 100mM after administration of the one or more compounds of Table 1. In someembodiments, the concentration of one or more compounds of Table 1 inthe brain is at least 10 μM after administration of the one or morecompounds of Table 1.

Accordingly, methods of treatment of JCV infection and PML may beevaluated prior to initiating treatment with an immunosuppressive agent,during administration of an immunosuppressive agent, or assessed afteran immunosuppressive agent has been administered or after treatment withimmunosuppressive treatment has been terminated.

As used herein, “diagnosing” and “evaluating treatment of PML” comprisesdetermining the presence of JCV infection. Determining the presence ofJCV infection comprises the detection of JCV in one or more tissues orin fluids (which can include determining the viral load in blood and/orcerebral spinal fluid) and may include determining the sequence of JCV.Diagnostic assays include but are not limited to histopathology,immunohistochemistry, flow cytometry, cytology, patho-physiologicalassays, including MRI and tomography, neurological assays biochemicalassays. Biochemical assays include but are not limited to variantanalysis, viral genome analysis, ELISA analysis, including the use ofantibodies against one or more proteins of JCV, analysis of specificproteins, platelet count, etc. Those of ordinary skill in the art willbe aware of numerous diagnostic protocols and parameters that areroutinely utilized in the art.

In some aspects of the invention, PML-specific diagnostic tests andmethods of monitoring PML symptoms may be used in connection withmonitoring and treating a subject with PML, a subject suspected ofhaving PML, a subject at risk for PML, a subject being treated withimmunosuppressants, and/or a subject currently under treatment for PML.PML diagnostic criteria may be used in the assessment of subjects beingtreated (e.g., to monitor efficacy of treatment) or to assist in thedecision whether to treat a subject with a method or composition of theinvention, including, but not limited to, whether to supplement analternative treatment with a treatment of the invention. In someembodiments, PML diagnostic criteria may be used to assist in thedecision regarding treatment of a subject with immunosuppressant, e.g.,to terminate, temporarily halt, or decrease the dose ofimmunosuppressant treatment regimens. Symptoms of PML are neurologicaland include problems with hand-eye coordination, including difficultywriting and typing, as well as problems with speech, hemiparesis andnonfluent aphasia. In some cases, an MRI scan showing an increase in theextent of the high T2-weighted and low T1-weighted signal abnormalitiescompared to a normal reference is indicative of PML. MRI may be used tomonitor changes in lesion size and progression of PML. Detection of JCVDNA by PCR in CSF is a widely used diagnostic test of PML, it has 99%specificity and 70% selectivity. Brain biopsy for detection of JCV DNAmay also be performed to diagnose or assess PML. In the absence of JCVPCR+ result for CSF, a brain biopsy may be performed and JCV DNAdetection in brain tissue is used as a positive diagnosis of PML. Kapposet al., Natalizumab treatment for multiple sclerosis: recommendationsfor patient selection and monitoring, Lancet Neurol., 2007 May, 6(5):431-41, describes non-limiting examples of diagnostic and managementalgorithms to monitor patients (e.g., multiple sclerosis patients) thatare treated with natalizumab. Patients can be monitored using acombination of clinical, MRI, and laboratory assessments. The algorithmsof Kappos et al. are incorporated herein by reference in their entirety.

As used herein, methods of the invention may be carried out in subjects.A subject may be a human or a non-human animal, including, but notlimited to a non-human primate, cow, horse, pig, sheep, goat, dog, cat,or rodent. In general, all embodiments described herein may be appliedto human subjects where appropriate.

In some embodiments, the methods of treatment of the invention compriseadministration of compositions of compounds of Table 1 in combinationwith candidate therapeutic compounds identified in screens for activityagainst DNA viral infection or PML. Candidate compounds that haveactivity against DNA viral infection or PML can be identified through avariety of screening methods including both in vitro screens and in invivo screens. In vitro screens encompass both biochemical and biologicalassays. Biochemical assays encompass assays that can determine thebinding of candidate therapeutic compounds to a specific target, e.g., aprotein or other macromolecule of the DNA virus. Biological assaysencompass cellular assays, which can for instance be based on the uptakeof a virus in a cell, the release of a virus from a cell, infectionrate, viral DNA replication (e.g., measured as a viral DNA replicationrate, an amount of viral DNA in a cell, or other measure of viral DNAreplication) etc., or any combination thereof. In general, the assayswill have a readout (like fluorescence) which allow for thedetermination of the potential therapeutic efficacy of a candidatetherapeutic compounds for the treatment of infection with a DNA virus orPML. In some embodiments, an assay mixture for testing a candidate agentcomprises a candidate agent. A candidate agent may be an antibody, asmall organic compound, or a polypeptide, and accordingly can beselected from combinatorial antibody libraries, combinatorial proteinlibraries, or small organic molecule libraries. Typically, pluralitiesof reaction mixtures are run in parallel with different agentconcentrations to obtain a different response to the variousconcentrations. Typically, one of these concentrations serves as anegative control, e.g., at zero concentration of agent or at aconcentration of agent below the limits of assay detection. Any moleculeor compound can be a candidate therapeutic. Non-limiting examples ofcandidate therapeutics are small molecules, RNA including siRNAs, DNAincluding aptamers, and proteins including antibodies and antibodyfragments. The invention also embraces candidate therapeutic compoundswith different modes of action. Candidate agents encompass numerouschemical classes, although typically they are organic compounds,proteins or antibodies (and fragments thereof that bind antigen). Insome general embodiments, the candidate agents are small organiccompounds, e.g., those having a molecular weight of more than 50 yetless than about 2500, for example less than about 1000 and, in certainembodiments, less than about 500. Candidate agents comprise functionalchemical groups necessary for structural interactions with polypeptidesand/or nucleic acids, and may include at least an amine, carbonyl,hydroxyl, or carboxyl group, optionally at least two of the functionalchemical groups or at least three of the functional chemical groups. Thecandidate agents can comprise cyclic carbon or heterocyclic structureand/or aromatic or polyaromatic structures substituted with one or moreof the above-identified functional groups. Candidate agents also can bebiomolecules such as nucleic acids, polypeptides, saccharides, fattyacids, sterols, isoprenoids, purines, pyrimidines, derivatives orstructural analogs of the above, or combinations thereof and the like.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides, synthetic organic combinatorial libraries, phagedisplay libraries of random or non-random polypeptides, combinatoriallibraries of proteins or antibodies, and the like. Alternatively,libraries of natural compounds in the form of bacterial, fungal, plant,and animal extracts are available or readily produced. Additionally,natural and synthetically produced libraries and compounds can bereadily be modified through conventional chemical, physical, andbiochemical means. Further, known agents may be subjected to directed orrandom chemical modifications such as acylation, alkylation,esterification, amidification, etc. to produce structural analogs of theagents.

A variety of other reagents also can be included in the mixture. Theseinclude reagents such as salts, buffers, neutral proteins (e.g.,albumin), detergents, etc., which may be used to facilitate optimalprotein-protein and/or protein-agent binding. Such a reagent may alsoreduce non-specific or background interactions of the reactioncomponents. Other reagents that improve the efficiency of the assay suchas protease inhibitors, nuclease inhibitors, antimicrobial agents, andthe like may also be used.

In some embodiments, candidate therapeutic compounds are based on thecompounds of Table 1 and comprise modified versions of the compounds ofTable 1 generated through methods of medicinal chemistry known to theskilled artisan.

In another aspect, the present invention provides “pharmaceuticalcompositions”, which comprise a therapeutically effective amount of oneor more of the compounds described herein, formulated together with oneor more pharmaceutically acceptable carriers (additives) and/ordiluents. As described in detail, the pharmaceutical compositions of thepresent invention may be specially formulated for administration insolid or liquid form, including those adapted for the following: oraladministration, for example, drenches (aqueous or non-aqueous solutionsor suspensions), tablets, e.g., those targeted for buccal, sublingual,and systemic absorption, boluses, powders, granules, pastes forapplication to the tongue; parenteral administration, for example, bysubcutaneous, intramuscular, intravenous or epidural injection as, forexample, a sterile solution or suspension, or sustained-releaseformulation; topical application, for example, as a cream, ointment, ora controlled-release patch or spray applied to the skin, lungs, or oralcavity; intravaginally or intrarectally, for example, as a pessary,cream or foam; sublingually; ocularly; transdermally; or nasally,pulmonary and to other mucosal surfaces.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, involved in carrying or transporting the subject compound fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides;and other non-toxic compatible substances employed in pharmaceuticalformulations.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, and the particular mode ofadministration. The amount of active ingredient that can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.Generally, this amount will range from about 1% to about 99% of activeingredient, for example from about 5% to about 70%, and in someembodiments from about 10% to about 30%.

In certain embodiments, a formulation of the present invention comprisesan excipient selected from the group consisting of cyclodextrins,liposomes, micelle forming agents, e.g., bile acids, and polymericcarriers, e.g., polyesters and polyanhydrides; and a compound of thepresent invention. In certain embodiments, an aforementioned formulationrenders orally bioavailable a compound of the present invention.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically-acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: fillers or extenders, such as starches, lactose, sucrose,glucose, mannitol, and/or silicic acid; binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; humectants, such as glycerol; disintegratingagents, such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; solutionretarding agents, such as paraffin; absorption accelerators, such asquaternary ammonium compounds; wetting agents, such as, for example,cetyl alcohol, glycerol monostearate, and non-ionic surfactants;absorbents, such as kaolin and bentonite clay; lubricants, such as talc,calcium stearate, magnesium stearate, solid polyethylene glycols, sodiumlauryl sulfate, and mixtures thereof; and coloring agents. In the caseof capsules, tablets and pills, the pharmaceutical compositions may alsocomprise buffering agents. Solid compositions of a similar type may alsobe employed as fillers in soft and hard-shelled gelatin capsules usingsuch excipients as lactose or milk sugars, as well as high molecularweight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made in asuitable machine in which a mixture of the powdered compound ismoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be formulated for rapid release,e.g., freeze-dried. They may be sterilized by, for example, filtrationthrough a bacteria-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions that can be dissolvedin sterile water, or some other sterile injectable medium immediatelybefore use. These compositions may also optionally contain opacifyingagents and may be of a composition that they release the activeingredient(s) only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions that can be used include polymeric substances andwaxes. The active ingredient can also be in micro-encapsulated form, ifappropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically-acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Dissolvingor dispersing the compound in the proper medium can make such dosageforms. Absorption enhancers can also be used to increase the flux of thecompound across the skin. Either providing a rate controlling membraneor dispersing the compound in a polymer matrix or gel can control therate of such flux.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containsugars, alcohols, antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers, which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms upon the subject compounds may be ensuredby the inclusion of various antibacterial and antifungal agents, forexample, paraben, chlorobutanol, phenol sorbic acid, and the like. Itmay also be desirable to include isotonic agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions, which are compatible with body tissue.

In certain embodiments, a compound or pharmaceutical preparation isadministered orally. In other embodiments, the compound orpharmaceutical preparation is administered intravenously. Alternativeroutes of administration include sublingual, intramuscular, andtransdermal administrations.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1% to 99.5% (incertain embodiments, 0.5% to 90%) of active ingredient in combinationwith a pharmaceutically acceptable carrier.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given in formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. In some embodiments, oral administrations are used.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically-acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular compound being employed, the duration ofthe treatment, other drugs, compounds and/or materials used incombination with the particular compound employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required to achievethe desired therapeutic effect and then gradually increasing the dosageuntil the desired effect is achieved.

In some embodiments, a compound or pharmaceutical composition of theinvention is provided to a subject chronically. Chronic treatmentsinclude any form of repeated administration for an extended period oftime, such as repeated administrations for one or more months, between amonth and a year, one or more years, or longer. In many embodiments, achronic treatment involves administering a compound or pharmaceuticalcomposition of the invention repeatedly over the life of the subject. Incertain embodiments, chronic treatments involve regular administrations,for example one or more times a day, one or more times a week, or one ormore times a month. In general, a suitable dose such as a daily dose ofa compound of the invention will be that amount of the compound that isthe lowest dose effective to produce a therapeutic effect. Such aneffective dose will generally depend upon the factors described above.Generally doses of the compounds of this invention for a patient, whenused for the indicated effects, will range from about 0.0001 to about100 mg per kg of body weight per day. In some embodiments, the dailydosage will range from 0.001 to 50 mg of compound per kg of body weight,for example from 0.01 to 10 mg of compound per kg of body weight.However, lower or higher doses can be used. In some embodiments, thedose administered to a subject may be modified as the physiology of thesubject changes due to age, disease progression, weight, or otherfactors.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

While it is possible for one or more compounds of the present inventionto be administered alone, in general embodiments one or more compoundsmay be administered as a pharmaceutical formulation (composition) asdescribed herein.

The compounds according to the invention may be formulated foradministration in any convenient way for use in human or veterinarymedicine, by analogy with other pharmaceuticals.

The invention also relates to a method of making a medicament for use intreating a subject, e.g., for treating or preventing a DNA virus (e.g.,JCV or BKV) infection, for inhiting a DNA virus replication orproliferation. Such medicaments can be used for prophylactic treatmentof a subject at risk for or suspected of having a DNA virus infection(e.g., for treatment of a subject prior to, during, and/or after thesubject receives an immunomodulatory therapy). Accordingly, one or morecompounds or compositions described herein that modulate DNA virusreplication or proliferation as described herein may be used for thepreparation of a medicament for use in any of the methods of treatmentdescribed herein. In some embodiments, the invention provides for theuse of one or more compounds or compositions of the invention (e.g.,identified as inhibiting DNA virus replication) for the manufacture of amedicament or pharmaceutical for treating a mammal (e.g., a human)having one or more symptoms of, or at risk for, DNA virus infection,replication and/or proliferation (e.g., one or more symptoms of JCV orBKV activity, or one or more symptoms of another DNA virus activity).Accordingly, aspects of the invention relate to the use of one or morecompounds or compositions of the invention for the preparation of amedicament for treating or preventing PML in a subject.

Accordingly, the invention also relates to one or more compounds orcompositions of the invention for use as a medicament. The inventionalso relates to one or more of these compounds or compositions for usein methods of the invention, for example in methods of inhibiting DNAvirus (e.g., JCV or BKV) replication, or of treating or preventing adisease associated with DNA virus replication or proliferation (e.g., insubjects that are about to be, are being, and/or have been treated withat least one immunomodulatory composition).

In some aspects, the invention provides kits comprising one or morecompounds of Table 1 and instructions for administering the one or morecompounds of Table 1. In some embodiments, the kit also comprises animmunosuppressant and instructions for administering the one or morecompounds of Table 1 and the immunosuppressants. In some embodiments,the immunosuppressant is natalizumab. The components of a kit can beincluded in a container or package having one or more positions for eachcomponent (and each component can be separately packaged in a dry,liquid, gel, or other form).

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting.

EXAMPLES Example 1 Detection of JCV and JCV Variants by PCR

Nucleic acids are isolated from a biological sample using establishedprotocols (e.g., cell lysis). Because the viral DNA may have integratedin the genomic DNA or may still be present as a smaller entity, bothgenomic DNA and shorter DNA sequences may be isolated and subjected toPCR analysis. Upon isolation the nucleic acids are resuspended in abuffer that will facilitate PCR analysis. Buffers that facilitate PCRanalysis are known to the skilled artisan and are also commerciallyavailable from manufacturers of PCR enzymes (e.g., New England Biolabs,Beverly, Mass.). Nucleotide primers are designed to result in theamplification of a JCV gene. PCR amplification is an establishedlaboratory technique and comprises the addition of nucleotide primers, apolymerase and single nucleotides, and polymerase buffer and subjectionthis mixture to cycles of annealing, amplification and dissociationresulting in the amplification of a desired DNA sequence. Uponamplification, the JCV gene is separated from the residual DNA andexcess single nucleotides. The amplified JCV DNA is sequenced and theresulting nucleotide sequence is translated into a peptide sequence todetermine if JCV polypeptides and polypeptide variants are present inthe biological sample.

Example 2 Detection of JCV and JCV Variants Using ELISA

Proteins and peptides are isolated from a biological sample usingstandard laboratory techniques. Both the cellular proteins and proteinsof non-cellular components can be subjected to the analysis. In oneassay the sample is interrogated for the presence of JCV polypeptides.The polypeptides are detected using sandwich ELISA comprising antibodiesspecific for JCV polypeptides. The antibodies are generated byinoculating animals (e.g., rabbits) with the JCV polypeptides of theinvention resulting in polyclonal antibodies. If so desired, cells canbe harvested from the inoculated animal to generate monoclonalantibodies. Methods for the generation of both polyclonal and monoclonalantibodies are routine in the art. The antibodies against JCVpolypeptide and JCV polypeptide variants are immobilized on a solidsurface (e.g., a 96-well plate), with one antibody type per well orsurface area. The biological samples comprising the polypeptides areadded to the wells and incubated with the immobilized antibodies. JCVpolypeptides and JCV polypeptide variants present in the sample willbind to an antibody specific for the polypeptide. After incubation, thesample is removed and the solid surfaces are washed to remove anyunbound material. As a next step, a solution containing additionalantibodies specific for JCV peptides is added to the wells. This secondaliquot of antibodies will create the “sandwich” (e.g., immobilizedantibody: JCV polypeptide: second antibody). This second antibody can bedetected using, for instance, a labeled tertiary antibody, allowing forthe detection of JCV variant polypeptides. Alternatively, the secondaryantibody itself may be labeled.

In a second ELISA assay, biological samples are assayed for the presenceof antibodies against one or more CV polypeptides or JCV variantpolypeptides. This assay can be use to determine whether a subject iscurrently infected with, or has previously been exposed to, JCV or a JCVvariant. Even if a specific JCV variant is no longer present, antibodiesagainst the variant may still be present in the biological sample andcan be detected. In this ELISA assay JCV polypeptides are attached to asolid surface and the biological samples are incubated with thesepolypeptides. If antibodies specific for these polypeptides are presentin the biological samples they will bind to the polypeptides. Anyunbound material is again removed. The presence of bound antibody isdetected using a labeled secondary antibody.

Example 3 Treatment of JCV Infection

SV40 transformed glial cells were seeded in 10% FBS media. The cellswere seeded at 2000 cells per well in 75 μl of media per well. On day 2,the media was removed and replaced by 35 μl of a 1× concentrated drugaliquot combined with a 100× diluted JCV Turbo aliquot in 35 μl of 2%FBS media (JCV Turbo is a hybrid Mad-1/SVEΔ virus constructed byinsertion of the regulatory region of SV40 into the regulatory region ofthe Mad-1 strain of JCV (Mad-1/SVE), Vacante et al., Virology 1989, 170:353-361). The cells were incubated for an hour, after which another 65uL aliquot of 1× concentrated drug in 2% FBS was added. On day 5 thecells were stained with DAPI (for total cell number) and with a mousemonoclonal antibody against SV40 VP1, which cross reacts with the JCVVP1 protein. The JCV VP1 protein is displayed on the cell surface when acell is infected by JVC1. The ability of the drug to suppress or inhibitJCV infection is shown in Table 2. The information is tabulated as %inhibition (as measured by the number and percentage of JCV positivecells). The cytotoxocity of the drug is displayed in the third column.

In a separate experiment the IC₅₀ was determined. FIG. 1 shows theinhibition curve for different cell seedings. The y-axis shows thepercentage inhibition depending on the concentration of neutralizingantibody added (Neutralizing antibody is a rabbit polyclonal fromanti-JCV neutralizing serum).

In a further experiment the correlation between concentration of JCV and% infection was determined. FIG. 2 shows that a JCV dilution of 1:50results in 6% of infected cells, while a JCV dilution of 1:500 resultsin a 1.5% infection.

TABLE 2 Inhibition of JCV infection % Inhibition % Inhibition (total %Inhibition total Cell Molecular #JCV+) (% JCV+) Number MOLENAME Weight@10 uM sd @10 uM sd @10 uM sd CHLOROACETOXYQUINOLINE 221.64276 38 0 20 220 2 DEMETHYLNOBILETIN 388.37384 38 0 20 2 20 2 PROPANIL 218.08202 12 1421 0 −12 17 AMINOETHOXYDIPHENYLBORANE 225.09822 25 5 21 12 3 85-NITRO-2- 300.31409 14 9 21 1 −9 9 PHENYLPROPYLAMINOBENZOIC ACID [NPPB]3beta- 414.62848 25 5 21 12 3 8 HYDROXYISOALLOSPIROST-9(11)- ENE LEOIDIN413.21021 14 9 21 1 −9 9 PICROPODOPHYLLOTOXIN 414.41168 19 4 22 1 −1 5THIABENDAZOLE 201.25182 19 4 22 1 −1 5 HARMANE 182.22488 24 0 22 8 4 86,4′−DIHYDROXYFLAVONE 254.242 18 0 22 6 −5 8 GENTIOPICROSIDE 356.3292932 5 22 3 11 2 (R)-ANGOLENSIN 272.30063 18 0 22 6 −5 8 PTAEROXYLIN258.27374 32 5 22 3 11 2 DIPYRIDAMOLE 504.63297 24 8 22 3 3 6 NABUMETONE228.29083 20 5 23 4 −2 1 ROSIGLITAZONE 357.43323 22 3 23 3 −1 8DILTIAZEM HYDROCHLORIDE 450.98602 20 1 24 0 −4 0 BETAMETHASONE 392.467534 0 24 4 14 4 ICHTHYNONE 408.40741 34 0 24 4 14 4 AMCINONIDE 502.579816 14 25 7 −11 9 RILUZOLE 234.2018 16 14 26 9 −12 5 FLUFENAMIC ACID281.23413 37 8 26 8 14 1 CHRYSIN 254.24199 18 5 26 8 −12 5 DICTAMNINE199.20902 35 1 26 3 12 5 PIPLARTINE 317.34152 21 11 27 2 −7 11 PEUCENIN260.28964 21 11 27 2 −7 11 METHOXYVONE 266.29636 38 1 27 2 15 4ISOTRETINOIN 300.44104 39 10 28 1 15 13 CHLOROXYLENOL 156.61157 36 2 2910 10 9 TOMATINE 994.13745 40 8 29 15 12 7 PRIMULETIN 238.24258 40 8 2915 12 7 MEFENAMIC ACID 241.28966 29 4 29 2 0 9 DIETHYLSTILBESTROL268.35556 42 10 29 13 18 1 CHLORAMPHENICOL PALMITATE 505.43799 20 5 29 9−13 6 METHYLXANTHOXYLIN 210.22975 33 3 30 1 5 2 L-ALANINOL 75.110596 132 31 3 −23 9 DICLOFENAC SODIUM 318.13409 44 2 31 1 18 4 FLUNIXINMEGLUMINE 491.46429 30 11 33 12 −4 2 DEHYDROABIETAMIDE 299.45636 40 4 337 11 3 PACHYRRHIZIN 336.30057 45 0 34 1 17 2 DICUMAROL 336.3006 36 13 3511 3 4 DIFFRACTIC ACID 374.39035 44 0 36 2 13 2 ACEMETACIN 415.82962 327 38 3 −8 6 GINKGOLIC ACID 346.51007 33 11 39 6 −8 8 XANTHONE 196.2053439 0 39 3 1 4 FUSIDIC ACID 516.7182 39 6 40 4 −2 16 POLYMYXIN B SULFATE1301.5724 36 3 41 8 −8 10 PYRANTEL PAMOATE 594.68781 48 2 42 0 12 34-(3-BUTOXY-4- 278.35132 48 2 42 0 12 3 METHOXYBENZYL)IMIDAZOLIDIN-2-ONE MICONAZOLE NITRATE 479.14554 46 1 42 1 7 1 CANDESARTAN CILEXTIL610.66943 54 5 44 4 19 3 ENDOSULFAN 406.92694 54 3 44 2 19 2DIOXYBENZONE 244.24689 55 8 49 8 11 1 TOLFENAMIC ACID 261.70752 61 2 530 18 5 MEFLOQUINE 378.3172 66 10 64 7 5 10

Example 4 Identification and Characterization of Mefloquine EfficacyAgainst JC Virus

In order to identify the drugs with anti-JCV activity, a commerciallyavailable collection of approved drugs and bioactive compounds werescreened in in vitro JC viral infection assay. As a primary screen,inhibition of the viral infection rate was monitored in human glial cellline SVG-A (Major, Miller et al. 1985) infected with JCV strainMad1/SVEΔ (Vacante, Traub et al. 1989). The infection rate was measuredas a percent of cells expressing viral envelop protein VP1 usingCellomics ArrayScan (Pittsburgh, Pa.). Out of 2000 compounds in theSPECTRUM collection screened, 14 were identified that had inhibited thenumber of infected cells by >50% at the concentrations <20 μM (IC₅₀<20μM). Since PML is a result of uncontrolled viral replication in the CNS,the compounds were evaluated to determine their ability to cross theblood brain barrier in sufficient concentration to be therapeuticallyeffective. Based on the published literature, mefloquine shows CNSpenetration that could be expected to achieve in vitro derivedefficacious concentrations in humans (Jones, Kunsman et al. 1994; Pham,Nosten et al. 1999).

Using qPCR to quantify the number of viral copies in the culture, italso was shown that mefloquine inhibits viral DNA replication. Furtherexperiments with mefloquine demonstrated its ability to inhibitinfection by another JCV strain, Mad4 and in a different cell type,primary human astrocytes. Mefloquine is similarly effective in theinhibition of JCV infection rate even when added to the culture system24 hrs after infection of cells with the virus, suggesting that itinhibits virus in previously infected cells. Both (+) and (−)enantiomers of mefloquine racemate were similarly potent at inhibitingJCV infection in the JCV inhibition assay.

Mefloquine hydrochloride is an antimalarial agent indicated for thetreatment and prophylaxis of mild to moderate acute malaria caused bymefloquine-susceptible strains of P. falciparum and P. vivax and has asignificant history of use in human population, with 11 million patientstreated since 1984, when it was first registered. Although no animalmodel of PML or JCV infection is available to test mefloquine in vivo,in vitro results and published literature show that mefloquine is aneffective anti-JCV therapy.

Materials and Methods

Compounds: The 2,000 compounds Spectrum Collection (MicroSourceDiscovery Inc., Groton, Conn.) consists of ˜1000 Drugs defined accordingto the name designations as set forth in the USP Dictionary of USAN andInternational Drug Names (2005, US Pharmacopeia), including Food andDrug Administration (FDA) approved drugs, and other bioactive compoundsand natural products. An alphabetical list of the compounds is availableat “The Spectrum Collection” internet site. The compounds are suppliedas 10 mM solutions in dimethyl sulfoxide (DMSO). Mefloquine waspurchased from Sigma (Sigma-Aldrich, St. Louis, Mo.). Two mefloquineenantiomers were separated from a commercial mefloquine sample by ChiralTechnologies (Chiral Technologies, West Chester, Pa.) using chiral HPLCon a CHIRALPAK IA column.

Cell Lines: The human glial cell line SVG-A (a gift from Walter Atwood),established by transformation of human fetal glial cells by anorigin-defective SV40 mutant (Major, Miller et al. 1985), was culturedin 1× Eagle Minimal Essential Media (MEM) supplemented with 10%heat-inactivated fetal bovine serum, 4 mM L-glutamine (Mediatech, HolyHill, Fla.). Infection was performed in 1× Eagle Minimal Essential Media(MEM) supplemented with 2% heat-inactivated fetal bovine serum, 4 mML-glutamine (Mediatech, Inc.). Human astrocytes (ScienCell ResearchLaboratories, San Diego, Calif.) were isolated from fetal cerebralcortex and cultured in proprietary basal medium, supplemented with 2%fetal bovine serum, 1% astrocyte growth, and penicillin/streptomycin(ScienCell Research Laboratories). Infection of astrocytes was performedin this media.

JC Polyoma Viruses: Hybrid Mad-1/SVEDelta virus (Mad-1/SVEΔ virus) (agift from Walter Atwood) was constructed by insertion of the regulatoryregion of SV40 into the regulatory region of the Mad-1 strain of JCV(Mad-1/SVE) (Vacante, Traub et al. 1989). Virus was propagated on SVG-Acells and purified as previously described (Liu, Hope et al. 1998).Briefly, SVGA cells plated at 50% confluence are infected with a 1:50dilution of the MAD1-SVE delta strain of JC virus for 1 hour at 37° C.That concentration of virus has been shown to give a maximal infectionrate of SVGA cells. Cells are cultured for 3 weeks with weekly changesof media. After 3 weeks of culture, cells are scraped from the flasks,pooled, including loose cells from prior media changes, and pelleted.The cell pellet is then resuspended in 20 ml of supernatant anddisrupted in a microfluidizer (Microfluidics inc., Newton, Mass.).Deoxycholate is added to a final concentration of 0.25% and incubated at37° C. for 30 minutes. The virus-containing cellular supernatant is thencentrifuged at 10,000 RPM for 30 minutes in a SA600 rotor. Thesupernatant is then aliquoted and stored at −80° C. The nonarchetypalstrain of JC, Mad4 (Major, Vacante et al. 1987; Frye, Trebst et al.1997) was obtained from ATCC (Manassas, Va.).

Detection antibodies: PAB597 (a gift from Walter Atwood), a mousemonoclonal to SV40 V antigen, cross-reacts to JCV VP1 (Atwood, Wang etal. 1995) and was used to visualize JC infection with secondarydetection using an Alexa-Fluor 488-labeled goat anti-mouse secondaryantibody (Molecular Probes). Cell nuclei were stained with4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, Calif.).Neutralizing anti-JCV rabbit antisera was a gift from Walter Atwood(Atwood 2001).

JCV Infectivity Assay: SVG-A cells were seeded at 2,000 cells/well/0.075ml of culture media in 96 flat-bottom well plate (Corning, N.Y.). Thenext day compounds were prepared in assay medium (2% heat-inactivatedfetal bovine serum, 4 mM L-glutamine, 1×MEM). A master viral plate wasprepared by mixing equal volumes of [2×] compound and [2×] diluted virusfor the final 1× concentration of the compound and the virus. The platecontaining cells was gently inverted and shaken to remove media. Fromthe master plate, 0.035 ml compound/virus was added to designated wells.The cells were incubated with the compound/virus mixture for 60 minutesin a humidified 37° C. CO₂ incubator. At that time final concentrationof the drug in media was added to designated wells to bring the finalvolume up to 0.1 ml/well. The plates were incubated for an additionalthree days, the cells were then washed once with 1×PBS, and fixed in 2%paraformaldehyde/1×PBS for 30 minutes at room temperature. The fixativewas removed and the cells were solubilized with 0.5% Triton X100 in PBSfor an additional 30 minutes. Infection by Mad-1/SVEA virus wasvisualized by staining with PAB597, a monoclonal antibody against themajor capsid protein VP1, the cells were incubated with 0.05 ml ofPAB597 (2 μg/ml in 1×PBS) for 60 minutes at 37° C. Following a wash stepwith 1×PBS, primary antibody was detected with an Alexa-Fluor488-labeled goat anti-mouse secondary antibody at 1:100 dilution in1×PBS, cells were counterstained with DAPI at 1 μg/ml (0.05 ml/well) for30 minutes at 37° C. Cells were washed with 1×PBS and 0.1 ml 1×PBS wasadded to cells. Field images of each well were acquired and analyzedusing the Cellomics ArrayScan (Thermo Scientific Inc, Waltham, Mass.)using the Target Activation software. Human astrocytes follow the samebasic assay protocol with some notably exceptions. The cells are seededat 4,000 cells/well/0.075 ml of culture media in 96 flat-bottom wellplate (Corning). The culture media is used for the infection. The lengthof the infection is six to ten days (instead of the three days for SVG-Acells).

Real time PCR: Taqman forward and reverse primers and MGB probes weredesigned for JC virus TAg using Primer Express v1.0 (Applied Biosystems,Foster City, Calif.) according to manufacture's recommendation. Tocreate a copy number standard curve for absolute quantification, pUC19plasmid containing JC virus genome was linearized using SmaI.Linearization was confirmed by capillary electrophoresis using anAgilent 12000 kit according to manufacturer's recommendation.Concentrations were determined by A₂₆₀ nm measurement on a nanodropspectrophotometer. Linearized plasmid was diluted 1/10 in TE starting at5×108/uL. Quadruplicate PCR reactions were run in a 384 well opticalplate (Applied Biosystems, Foster City, Calif.). Real time reactionswere cycled in a 7900HT (Applied Biosystems, Foster City, Calif.)thermal cycler under the following conditions: 50° C. for 2 minutes(uracil N-deglycosylase digest), 95° C. 10 minutes (activation of Taqthermostable polymerase), and 40 cycles of 95° C. for 15 seconds and 60°C. for 60 seconds with 900 nM forward and reverse primers, 200 nM Taqmanprobe, and 1× Universal master mix (Applied Biosystems, Foster City,Calif.). The fluorescence emission was collected every seven seconds forthe length of the run for each reaction well. Copy number was determinedfor each experimental sample by comparison to the absolute JC plasmidstandard curve using Sequence Detection Software (Applied Biosystems,Foster City, Calif.). The adjusted copy number was calculated by firstsubtracting the experimentally determined mass of JC virus DNA from thetotal DNA mass that was added to each 20 ul rxn well. P-values werecalculated using a Student t-test. JCV copy number was quantified bycomparison to a standard and normalized by the total DNA extracted froma sample. Zero copies of JC virus were detected in a non-infectednegative control.

DNA extraction and sample prep: DNA was extracted using to QIAamp 96blood kit (cat#51161; Qiagen Inc., Valencia, Calif.) with optional RNaseA treatment. DNA was quantitated using Quant-iT dsDNA high sensitivityassay according to manufacturer's recommendations (cat # Q33120Molecular Probes Inc., Eugene, Oreg.). Purified DNA was stored at −20°C. until use.

Calculations: The rate of JCV infection was calculated by normalizingthe number of JCV infected cells by the total number of nucleated cellsas % JCV⁺ cells=((total # VP1⁺ cells)/(total # DAPI⁺ cells))*100%. The %of viral inhibition by a compound was calculated using a JCV infectionrate instead of using total number of JCV infected cells (e.g., VP1⁺cells). Percent JCV Inhibition=100%*(1−(% JCV⁺ cells with a compound−%JCV⁺ cells in positive control)/(% JCV⁺ cells in positive control−% JCV⁺cells in negative control). The positive control is the cells infectedwith the virus in the absence of any compound, negative control are thecells not infected with any virus. The number of JCV⁺ cells in thenegative control samples quantitated by Cellomics ArrayScan were always<1% of the number of JCV⁺ in the positive control. The percent of JCVDNA inhibition was calculated as 100%*(1−(JCV copy# with a compound−JCVcopy# in positive control)/JCV copy# in positive control. Zero copies ofJCV genome were detected in no-infected negative control samples. Forhigh throughput screening of compounds, theZ-factor=1−3*((σp+σn)/∥μp−μn|; the mean (μ) and standard deviation (σ)of both the positive (p) and negative (n) controls (Zhang, Chung et al.1999) were calculated. Intraplate intragroup CV was always below 20% andZ′>0.5. IC₅₀ values were calculated using Prism software (GraphPadSoftware Inc., USA).

Primary Screen: JC Viral Infection Assay

In order to identify the drugs with anti-JCV activity, a commerciallyavailable collection of approximately 2000 approved drugs and bioactivecompounds called the SPECTRUM collection were screened for anti-JCVactivity in an in vitro viral infectivity assay (Pho, Ashok et al.2000). As a primary screen, the inhibition of the viral infection ratewas monitored in a human fetal astroglial cell line (SVG-A) infectedwith the JCV strain Mad1/SVEΔ. The SVG-A cell line (Major, Miller et al.1985) was chosen for the primary screening assay, as it is one of thefew available cell lines permissive for JC viral replication. Mad1/SVEΔJCV strain is a wild type Mad-1 virus originally isolated from the PMLpatient that had its regulatory non-coding region replaced with SV40regulatory region (Vacante, Traub et al. 1989). That insertion had beendemonstrated to extend species and cell-type host range of the virus.Infection of SVG-A cells with Mad1/SVEΔJCV strain allows for very fastviral replication and detection of virally infected cells within 3 daysof infection unlike 6-15 days need with other cell types and viralstrains.

Uniformity of a cell line, consistent infection rate and relativelyshort assay time are all important factors in creating a robust assayfor screening of a large number of compounds. To facilitate screening ofthe large compound collection, this JCV infectivity assay (Pho, Ashok etal. 2000) was used in a 96-well format and a Cellomics ArrayScan wasused to measure JCV replication. FIG. 3 shows the detection andmeasurement of cellular infection with JCV. In FIG. 3A SVG-A cellsinfected with JCV strain Mad1/SVEΔ 3 days earlier were fixed and stainedwith murine monoclonal antibodies specific for VP1 protein (greenstaining). The total cells present in the culture were visualized withDAPI DNA nuclear staining (blue) and picture was taken with CellomicsARRAYSCAN® camera at ×20 magnification. FIG. 3B illustrates the numberof infected cells (e.g., VP1⁺ cells) per group plotted against thedilution factor of the viral stock used to infect the cells (mean±SD,n=2). The total number of cells (bars) is similar for all groups. FIGS.3C and 3D illustrate results for cells that were infected in thepresence of various dilutions of JCV neutralizing antiserum orcidofovir. Three days later cells were fixed; stained and total numberof VP1⁺ cells and DAPI⁺ events per treatment group was enumerated usingCellomics ARRAYSCAN®.

In this system infected cells can be identified by immunofluorescentstaining with antibodies specific for JCV capsid protein VP1. Totalnumber of cells in culture was visualized via staining with DNA stainDAPI (FIG. 3A). ARRAYSCAN® was used to identify and count every singleevent in the assay well, routinely counting 300-800 VP1⁺ cells and8,000-16,000 DAPI⁺ events per well in a 96-well plate, thus minimizingvariability due to non-uniform cell growth pattern and/or intrawellviral spread and producing a very consistent result. As can be seen fromFIG. 3B, the number of infected cells (e.g., VP1⁺ cells) at the end ofculture period is proportional to the number of infectious viralparticles used to infect the cell culture. In these experiments, thehighest dilution of the viral stock that was convenient for applicationwas used. Using neutralizing rabbit anti-JCV serum as a positive controlfor viral inhibition it also was shows that the assay responds to viralinhibition in a predictive fashion (FIG. 3C).

During screening of the library for antiviral activity at single dose(10 μM), it was discovered that some of the compounds that mostdramatically inhibited the number of virally infected cells (e.g., VP1⁺cells) also dramatically reduced the total number of cells (e.g., DAPI⁺events) likely due to their cytotoxic/cytostatic effects. To determinewhether a particular compound had decreased the number of virallyinfected cells not just due its cytotoxic effect but due to itsantiviral effect, the percent of viral inhibition by a compound wascalculated using the JCV infection rate (e.g., % JCV⁺ cells=((total #VP1⁺ cells)/(total # DAPI⁺ cells))*100%) rather than total number of JCVinfected cells (e.g., the total number of VP1⁺ cells). In this systemJCV infection leads to 4-7% of all cells being infected in 72 hours. Ascan be seen from the example of treatment with cidofovir, a drug testedfor efficacy against PML (FIG. 3D), while it inhibited the number ofinfected cells in culture they also inhibited the total number of cellsin culture to the same degree so the percent inhibition of infectionrate (e.g., % JCV⁺ cells) was not significant. Similar effect wasobserved with other drugs with well reported cytotoxic effects, e.g.,Mytomicin C and cytarabine (data not shown).

Drug Screening and Selection

Following screening the library at a single compound concentration of 10μM, a number of drugs and compounds that effectively inhibited JCVinfection rate by >20% without significant cell toxicity (<20% totalcell number inhibition) were identified (FIG. 4). A level of 20% waschosen as a cut off for the first pass screening because the CV of theassay was consistently <20%. 67 compounds matched those criteria(Table 1) and were subsequently tested in the same assay across a fulldose response curve to further evaluate their therapeutic potential.Based on the results from full dose curves 14 drugs proved to beeffective, demonstrating >90% inhibition of the virally infected cells(IC₅₀<20 μM) without statistically significant cell toxicity (<20% totalcell number inhibition) at those concentrations (Table 3). Only thedrugs that did not reduce total cell numbers were selected in order todiminish the chance of confounding an anti-viral effect of the drug withits cytotoxic/cytostatic effect. The compounds that had reduced totalcell number by >80% were retested at the lower concentrations, but nonewere identified that demonstrated clear anti-JCV effect withoutconcomitant cytotoxic/cytostatic effect (FIG. 4). FIG. 4 shows the flowchart for SPECTRUM collection screening. The primary assay employed anSVG-A cell line and JCV Mad1/SVEΔ viral strain. The assay was performedas described for FIG. 3 (*IC₅₀-inhibition of % JCV infected cells by50%; TC₅₀-inhibition of total cell numbers by 50%).

The published literature was reviewed for information onpharmacokinetics and brain distribution of these drugs in humans and inanimal models. Since JC virus uncontrollably replicates in theoligodendrocytes and astrocytes of the effected individuals during PMLbut not all drugs are capable of crossing blood brain barrier it wouldbe advantageous for any potential PML therapy if the drug candidate iscapable of achieving efficacious concentration in the brain. Based onthe published literature (Table 3), mefloquine had been demonstrated toaccumulate in the brains of treated patients at the level of its invitro efficacious concentration (FIG. 3A; IC₅₀=3.9±2.1 μM). Brainconcentration of mefloquine based on postmortem brain analysis of peopletaking the drug prior to their deaths was 35-50 nmol per gram of braintissue, which could be approximated as 35-50 μM (Jones, Kunsman et al.1994; Pham, Nosten et al. 1999). This indicates that potentiallyefficacious doses of mefloquine can be achieved in the brain patientswho would be receiving approved doses of the drug.

Characterization of Mefloquine Activity in Primary Cell Culture

In order to further characterize the effect of mefloquine in JC virusinfectivity, experiments were performed to evaluate whether the JCVinhibitory effect of mefloquine was dependent on the cell line used inthe primary screen. Since SVG-A cells is a cell line propagated in vitrofor many generations, and is transformed with SV40 large T antigen thatcan enhance JCV replication, experiments were performed to evaluatewhether mefloquine is capable of inhibiting viral replication in cellsthat more closely resemble JCV target in human brain and do not expressSV40 TAg. Since in vitro infection culture of primary oligodendrocytes,a primary JCV target during PML, is not established, the JCV infectionof human fetal astrocytes was used to test the ability of mefloquine toinhibit the viral infection in primary cell culture. FIG. 5 shows theefficacy of mefloquine against two different viral strains (Mad1 andMad4) in two different cell types (SVG-A and primary astrocytes). FIG.5A illustrates SVG-A cells with Mad1/SVEΔ JCV (n=12) virus. FIG. 5Billustrates primary human fetal astrocytes with Mad1/SVEΔ JCV. FIG. 5Cillustrates SVG-A cells with JCV strain Mad-4 (n=5). % JCV Inhibitionwas calculated as 100%*(1−(% JCV⁺ cells with a compound−% JCV⁺ cells innegative control)/(% JCV⁺ cells in positive control−% JCV cells innegative control). Inhibition of total cell numbers (e.g., DAPI events)was less than 20% for all drug concentrations plotted. Unless otherwisenoted, one representative graph out of the number presented on the graphis shown. IC₅₀ are calculated as an average of all those experiments. Ascan be seen from FIG. 5A, mefloquine inhibits JCV infection of primaryhuman fetal astrocytes with essentially the same efficacy as it inhibitsviral infection in SVG-A cells. This result shows that anti-JCV effectof mefloquine is not dependent on cell type being infected by the virus,and that mefloquine is effective at inhibiting the virus in its“natural” setting of glial cell infection.

Characterization of Mefloquine Activity on Different JC Viral Strains

Experiments were also performed to determine whether the inhibitoryeffect of mefloquine is observed with other JC viral strains, and is notlimited to Mad1/SVEΔ JCV strain used in the primary screening assay. Theprimary screen was conducted with the Mad1/SVEΔ JCV strain because ofits fast replication in tissue culture. However, this viral straincontains a transcription regulatory region (non-coding) from itspolyomavirus family member SV40. To ensure that mefloquine's anti-viralactivity is not limited only to the virus with this modification,mefloquine's ability to inhibit an unmodified JCV strain, wild typevirus Mad4 that was originally isolated from a PML patient (Major,Vacante et al. 1987; Frye, Trebst et al. 1997), was tested. As can beseen from FIG. 5C, based on the results from 5 independent experiments,mefloquine inhibits Mad4 infection of SVG-A cells with the same efficacyas Mad1/SVEΔ infection. This result demonstrates that anti-JCV effect ofmefloquine does not depend on the viral strain.

Effect on JCV Viral DNA Replication

In order to better understand the mechanism of action of mefloquine andaddress whether this drug inhibits rate of cell infection by JC virusvia inhibition of viral DNA replication, viral DNA was quantified usingqPCR and a probe set specific for JCV T Antigen. As can be seen fromFIG. 6, the percent of JCV DNA inhibition by mefloquine almost overlapsthe percent of infection rate inhibition. A linear correlation betweenthe viral DNA copy number and a number of virally infected cells wasobserved (data not shown). This result demonstrates that mefloquineinhibits infection rate via its inhibition of viral DNA replication, andnot VP1 protein expression. FIG. 6 shows the effect of mefloquine on JCVDNA replication. SVG-A cells were infected with Mad1/SVEΔ JCV 3 daysearlier in the presence of various drug concentrations. % JCV DNAinhibition (⋄) was calculated using JCV copy numbers determined by qPCRwith a probe set specific for TAg. Inhibition of infection rate (x) wasmeasured in a replicate plate as described herein.

Effect of Mefloquine on Established JCV Infection

Experiments were performed to demonstrate that mefloquine is effectiveagainst established JCV infection. While it is apparent from the aboveexperiments that mefloquine is effective at inhibiting JCV infectionwhen added at the same time as the virus, it was not clear from thoseexperiments whether mefloquine inhibited viral entry into the cells, orthe viral life cycle in the cell. Since during PML many cells arealready infected with JC virus, in some embodiments a drug candidate fortreatment of PML may be selected as one that can inhibit viral lifecycle in already infected cells. As seen from FIG. 7, when added in 3 or24 hours post infection, mefloquine was just as effective at inhibitingcell infection as it was when added to the cells together with thevirus. Since most of the virus enters the cells within 1 hour, and allof the virus enters the cells within first 24 hours after infection(unpublished data), this result shows that mefloquine is effective ininhibiting viral replication in already infected cells. In FIG. 7various concentration of the drug were added to the culture of primaryhuman fetal astrocytes infected with JC virus at the same time as virus(circles; T=0), 3 hours after virus addition (squires; T=3 h), or 24hours after (triangles; T=24 h). Ten days after infection with the viruscells were fixed and number of virally infected cells was enumerated.Results of a representative experiment out of 4 different experimentswith primary astrocytes or SVG-A cells are shown.

Characterization of Mefloquine Enantiomers

Mefloquine is a racemic mix of (+) and (−) enantiomers of (R*,S*)-α-2-piperidinyl-2,8-bis (trifluoromethyl)-4-quinolinemethanolhydrochloride (FIG. 8). FIG. 8 shows the efficacy of different isomersof mefloquine. FIG. 8A shows IC₅₀s and structure of (R,S) and (S,R)enantiomers constituting drug racemate commercial mefloquine. (+) and(−) enantiomers were separated from a racemate via chiral chromatographyand added to SVG-A cells simultaneously with JCV strain Mad1/SVEΔ 3 daysearlier. After fixation and staining total number of VP1⁺ cells andDAPI⁺ events per treatment group was enumerated using CellomicsARRAYSCAN®. A representative experiment out of six performed is shown.While there is a minimal difference between the activities of theenantiomers against malaria (Basco, Gillotin et al. 1992; Brocks andMehvar 2003), the (−) enantiomer is much more potent in antagonisticactivity against A2a receptor (Weiss, Benwell et al. 2003). The twoenantiomers also display different pharmacokinetics and brainpenetration properties (Bourahla, Martin et al. 1996; Baudry, Pham etal. 1997). In order to better understand the anti-JCV effects of thesetwo components of the marketed mefloquine racemate, each enantiomer froma racemate was separated using chiral chromatography and compared themin a JCV inhibition assay. As can be seen from FIG. 8, both enantiomershave very similar efficacy in inhibiting JC virus. FIG. 9 shows theefficacy of different isomers of mefloquine and2,8-Bis-trifluoromethyl-quinolin. The compounds were added to SVG-Acells simultaneously with JCV strain Mad1/SVEΔ 3 days earlier. Afterfixation and staining total number of VP1⁺ cells and DAPI⁺ events pertreatment group was enumerated using Cellomics ARRAYSCAN®. Arepresentative experiment out of six performed is shown. The illustratedcompounds all inhibited JC virus replication. S,S-mefloquine andR,R-mefloquine have similar IC₅₀ similar to R,S-mefloquine andS,R-mefloquine, while a structurally related compound that has aaromatized ring has a slightly higher IC₅₀. FIG. 10. shows thatmefloquine anti-JCV activity is not inhibited by cerebrospinal fluid(CSF).

Based on this result and on the reported brain concentration formefloquine enantiomers (Baudry, Pham et al. 1997) it can be concludedthat mefloquine can be administered at concentrations efficacious in JCVinhibition in the brain of patients. Furthermore, lack of significantdifferences between anti-JCV activities of the enantiomers implies thatthis effect is not mediated by A2a receptor.

Mefloquine has been identified as a potential treatment for PML based onthe results of screening a commercially available collection of approveddrugs and bioactive compounds in in vitro JC viral infection assay.Follow-up experiments demonstrated that the effect of mefloquine is notlimited to one cell type or a single viral strain. Furthermore,mefloquine is capable of inhibiting viral replication in cells alreadyinfected with the virus. Both (+) and (−) enantiomers of mefloquineracemate were almost equipotent at inhibiting JCV infection, indicatingthat isolating individual enantiomers would not improved the benefit ofthe existing drug. In addition, the literature shows that mefloquinecrosses the blood brain barrier and can accumulate in the brain at thein vitro defined efficacious concentration for inhibition of JC viralinfectivity. Although no animal model is available for PML, the in vitroresults and literature data show that mefloquine is an effectiveanti-JCV therapy.

TABLE 3 Selected inhibitors (anti-JCV IC50 ≦20 μM, Therapeutic index(IC50/TC50) <0.5) TC₅₀, IC₅₀, Brain, plasma References for MOLENAMETHERAPY STATUS μM μM μM Cmax, μM PK data ISOTRETINON antiacne, USP, INN,ND 4.4 1100 ng/ml (Clamon, antineoplastic BAN Chabot et al. 1985;Colburn and Gibson 1985) MEFLOQUINE antimalarial USAN, INN, 16.1 4.030-50 6.0 (Jones, Kunsman BAN et al. 1994; Pham, Nosten et al. 1999)DICLOFENAC antiinflammatory USP, JAN 30.5 8.3 SODIUM DILTIAZEM Ca USP,INN, >40 8.5 HYDROCHLORIDE channel BAN, JAN blocker FUSIDICantibacterial USAN, INN, ND 8.6 1 μg/ml 10-90 μg/ml (Mindermann, ACIDBAN Zimmerli et al. 1993; Turnidge 1999) MICONAZOLE antifungal USP, JAN22.9 8.6 nd 10 ng/ml (Stevens, Konsil NITRATE (topical) et al. 2002)MEFENAMIC antiinflammatory, USP, INN, ND 10.9 0.8 μg/ml# 10-20 μg/ml(Glazko 1966; ACID analgesic BAN, JAN Fukuda, Kitaichi et al. 2005)FLUNIXIN analgesic, USP, 47.7 16.6 MEGLUMINE antiinflammatoryveterinarian PROPANIL herbicide >40 7.8 DEHYDROABIETAMIDE NA >40 13.0DIFFRACTIC NA >40 14.4 ACID HARMANE NA >40 14.4 XANTHONE NA ND 16.8METHOXYVONE NA >40 17.2 #animal data USP, United States PharmacopeiaINN, International Nonproprietary Name BAN, British Approved Name JAN,Japanese Approved Name Clamon, G., G. G. Chabot, et al. (1985). “Phase Istudy and pharmacokinetics of weekly high-dose 13-cis-retinoic acid.”Cancer Res 45(4): 1874-8. Colburn, W. A. and D. M. Gibson (1985).“Isotretinoin kinetics after 80 to 320 mg oral doses.” Clin PharmacolTher 37(4): 411-4. Fukuda, M., K. Kitaichi, et al. (2005). “Alteredbrain penetration of diclofenac and mefenamic acid, but notacetaminophen, in Shiga-like toxin II-treated mice.” J Pharmacol Sci97(4): 525-32. Glazko, A. J. (1966). “Experimental observations onflufenamic, mefenamic and meclofenamic acids. 3. Metabolic disposition.”Ann Phys Med Suppl: 23-36. Jones, R., G. Kunsman, et al. (1994).“Mefloquine distribution in postmortem cases.” Forensic Sci Int 68(1):29-32. Mindermann, T., W. Zimmerli, et al. (1993). “Penetration offusidic acid into human brain tissue and cerebrospinal fluid.” ActaNeurochir (Wien) 121(1-2): 12-4. Pham, Y. T., F. Nosten, et al. (1999).“Cerebral uptake of mefloquine enantiomers in fatal cerebral malaria.”Int J Clin Pharmacol Ther 37(1): 58-61. Stevens, R. E., J. Konsil, etal. (2002). “Bioavailability study of a 1200 mg miconazole nitratevaginal ovule in healthy female adults.” J Clin Pharmacol 42(1): 52-60.Turnidge, J. (1999). “Fusidic acid pharmacology, pharmacokinetics andpharmacodynamics.” Int J Antimicrob Agents 12 Suppl 2: S23-34.

Example 5 Identification and Characterization of Compounds that areActive Against JC Virus

FIG. 11 shows the dose response of several compounds that have anti-JCVactivity. FIGS. 12 and 13 show selected arylalkanoic acid NSAIDs andtheir anti-JCV activity. FIG. 14 shows the result of modeling studieswith mefloquine and related compounds and the IC₅₀s of a number ofcompounds structurally related to mefloquine. A pharmacophore-basedalignment method Catalyst and shape-based alignment method ROCS wereused to study the similarity between the compounds. The overlays below(or above) were derived in ROCS using scoring scheme that combines shapeand weighted chemical force-field overlap between molecules. Similarrelationships between molecules were detected through pharmacophorematching.

Table 4 shows the anti-JCV activity of a number of structural analogs tomefloquine (See also FIG. 15). Table 5 shows the anti-JCV activity of anumber of structural analogs to fusidic acid (See also FIG. 16).

It should be appreciated that any one or more of the structural analogsdescribed herein and/or any one or more of the compounds listed inTables 1-5 can be used according to methods of the invention if theyhave suitable properties as described herein.

TABLE 4 Selected compounds from mefloquine functional fingerprint insilico screen anti- JCV activ- Structure MOLREGNO MDLNUMBER MOLNAME CAS#ity Nc1ncnc2n(c(nc12)Cl)C1OC(CO)C(O)C1O 129461 MFCD010764578-CHLOROADENOSINE 34408-14-5 Yes Nc1nccc2n(cnc12)C1OC(CO)C(O)C1O 64582MFCD00153951 3-DEAZAADENOSINE 6736-58-9 Yes Nc1nc2ncnc2c(n1)OCC1CCCCC1289926 MFCD05664734 O6-Cyclohexylmethylguanine YesCCC(CO)Nc1nc(c2ncn(c2n1)C(C)C)NCc1ccccc1 182843 MFCD02266402ROSCOVITINE, (S)-ISOMER Yes Nc1ncnc2n(cnc12)C1OC(CO)C(O)C1O 5424MFCD00005752 ADENOSINE 58-61-7 No NC1═Nc2n(cnc2C(═O)N1)C1OC(CO)C(O)C1O9593 MFCD00010182 GUANOSINE 118-00-3 No CC(C)C(═O)c1c2n(nc1C(C)C)C═CC═C2123831 MFCD00864808 IBUDILAST 50847-11-5 NoOCC1OC(C(O)C1O)n1cnc2c(ncnc12)NCc1ccccc1 5414 MFCD000057406-BENZYLAMINOPURINE 4294-16-0 NA RIBOSIDENc1c2ncn(c2nc[n+]1[O—])C1OC(CO)C(O)C1O 21536 MFCD00037993ADENOSINE-N′-OXIDE NA NC1═Nc2n(cnc2C(═O)N1)C1OC(CO)C(O)C1O 38045MFCD00064103 BETA-L-GUANOSINE NAOCC1OC(C(O)C1O)n1cnc2c(ncnc12)NC1CC2CCC1C2 40907 MFCD00069271(2S)-N6-[2-ENDO- NA NORBORNYL]ADENOSINE Cn1cnc2c(ncnc12)NC1CCCC1 64492MFCD00153844 N-0840 NA Cc1cc(c2nccc(c2c1)C)C 74766 MFCD00191505 4,6,8-NA TRIMETHYLQUINOLINE Nc1nc2ncnc2c(n1)OCc1ccccc1 91708 MFCD00269931O6-BENZYLGUANINE 19916-73-5 NA Cc1cc(c2cccc(c2n1)C)C 93735 MFCD002723312,4,8- 18441-61-7 NA TRIMETHYLQUINOLINECN1C(═O)N(C)c2ncn(c2C1═O)CC1OCCO1 123874 MFCD00865218 DOXOFYLLINE69975-86-6 NA CC(C)Cn1cnc2c(nc3ccccc3c12)N 124036 MFCD00866946 IMIQUIMOD99011-02-6 NA CCCCN1C(═O)N(CCCC)c2ncn(c2C1═O)CC(C)═O 124063 MFC00867152DENBUFYLLINE 57076-71-8 NA CN1N═C(N)c2cn(c3ncnc1c23)C1OC(CO)C(O)C1O124573 MFCD00932413 6-AMINO-4-METHYL-8- 35943-35-2 NA(BETA-D-RIBOFURANOSYL)- 4H,8H-PYRROLO[4,3,2- DE]PYRIMIDO[4,5-C]PYRIDAZINE N(c1ccccc1)C1═CC(═Nc2ncnn21)c1ccccc1 124680 MFCD00951199BUTTPARK 57\40-37 NA CC(C)n1cnc2c(nc(nc12)NCCCO)NCc1ccccc1 280770MFCD04974145 6-BENZYLAMINO-2-(3- NA HYDROXYPROPYLAMINO)-9-ISOPROPYLPURINE OC(═O)c1cc2cc(c(cc2c(n1)C(═O)c1ccc(c(c1)O)O)O)O 280811MFCD04974186 1-(3′,4′- NA DIHYDROXYBENZOYL)-6,7- DIHYDROXYISOQUINOLINE-3-CARBOXYLIC ACID

TABLE 5 Selected fusidic acid analogs anti- JCV activ- StructureMOLREGNO MDLNUMBER MOLNAME ity CC(═O)OC1CCC2C3CCC4═CC(O)CCC4C3CCC12C93067 MFCD00271609 4-ESTREN-3- Yes BETA, 17- BETA-DIOL 17-ACETATECC(OC(C)═O)C1CCC2C3CC(O)C4CC(O)CCC4(C)C3CCC12C 93343 MFCD002718965-BETA- Yes PREGNAN- 3-ALPHA, 6-ALPHA, 20-BETA- TRIOL 20- ACETATECC(OC(C)═O)C1CCC2C3CCC4═CC(O)CCC4(C)C3CCC12C 93383 MFCD002719374-PREGNEN- Yes 3-BETA, 20- BETA-DIOL 20-ACETATECC(OC(C)═O)C1CCC2C3CC(O)C4CC(O)CCC4(C)C3CCC12C 93342 MFCD002718955-BETA- No PREGNAN- 3-ALPHA, 6-ALPHA, 20-ALPHA- TRIOL 20-ACETATECC(CC(O)═O)C1CCC2C3C(CC4CC(O)CCC4(C)C3CC(OC(C)═O)C12C)OC(C)═O 234323MFCD03695615 23-NOR-5- No BETA- CHOLANIC ACID-3- ALPHA, 7- ALPHA, 12-ALPHA- TRIOL 7, 12-DIACE- TATE

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The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

All publications, patents and sequence database entries mentionedherein, including those items listed below, are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

1-42. (canceled)
 43. A method comprising administering a compositioncomprising fusidic acid to a subject before, during or after the subjectreceives an immunomodulatory therapy for a disease or condition.
 44. Themethod of claim 43, wherein the composition is administered to thesubject during the immunomodulatory therapy.
 45. The method of claim 43,wherein the composition is administered to the subject before theimmunomodulatory therapy.
 46. The method of claim 43, wherein thesubject is known to have been exposed to a polyomavirus infection. 47.The method of claim 46, wherein the subject is identified as having beenexposed to a polyomavirus infection by detecting one or more serumantibodies against a polyomavirus molecule or by detecting one or morepolyomavirus molecules obtained from the subject.
 48. The method ofclaim 46, wherein the polyomavirus is JC virus or BK virus.
 49. Themethod of claim 46, wherein the subject has a polyomavirus infection.50. The method of claim 43, wherein the disease or condition is multiplesclerosis, rheumatoid arthritis, myasthenia gravis, systemic lupuserythematosus or an inflammatory bowel disease or syndrome.
 51. Themethod of claim 50, wherein the disease or condition is multiplesclerosis.
 52. The method of claim 50, wherein the disease or conditionis Crohn's disease.
 53. The method of claim 43, wherein theimmunomodulatory therapy comprises administration of a VLA-4 antibody.54. The method of claim 53, wherein the VLA-4 antibody is natalizumab.55. A method comprising administering a composition comprising fusidicacid to a subject who has a weakened immune system.
 56. The method ofclaim 55, wherein the subject has a polyomavirus infection.
 57. Themethod of claim 56, wherein the polyomavirus is JC virus or BK virus.58. A method comprising administering a composition comprising fusidicacid to a subject having multiple sclerosis, wherein the composition isadministered to the subject before, during or after the subject receivesan immunomodulatory therapy for multiple sclerosis.
 59. The method ofclaim 58, wherein the subject has a JC virus infection or a BK virusinfection.
 60. The method of claim 58, wherein the immunomodulatorytherapy comprises the administration of a VLA-4 antibody.
 61. The methodof claim 60, wherein the VLA-4 antibody is natalizumab.